Recent Publications

 

Li Y, Stafford WF, Hesselberg M, Hayes D, Wu Z, Byrne M.

Characterization of the self-association of human interferon-α2b, albinterferon-α2b, and pegasys.

J Pharm Sci. 2012 Jan;101(1):68-80.

 

The self-association of human interferon-α2b (hIFN-α2b), albinterferon-α2b (a recombinant protein with human serum albumin and hIFN-α2b peptides fused together in a single polypeptide chain), and Pegasys (PEGylated hIFN-α2a) was characterized by analytical ultracentrifugation analyses. By examining the apparent sedimentation coefficient distribution profiles of each protein at different concentrations, it was concluded that the above three proteins are self-associating in albinterferon-α2b formulation buffer. By model fitting of sedimentation data using SEDANAL software, the stoichiometry and equilibrium constants of the self-association of these proteins were characterized. The self-association of hIFN-α2b results in the formation of stable dimers, fast-reversible tetramers, octamers, and hexadecamers. In contrast, although both albinterferon-α2b and Pegasys are self-associated, their self-association stoichiometries are significantly different from that of hIFN-α2b. The self-association of albinterferon-α2b results in the formation of reversible dimers and trimers, whereas the self-association of Pegasys gives only reversible dimers. The self-association behaviors of hIFN-α2b and albinterferon-α2b involves attractive electrostatic forces, which can be suppressed to a negligible level in low pH (pH 4.0-4.5) and high salt concentration (400 mM NaCl) buffer, allowing quantification of their size variant contents by sedimentation velocity analysis.

 

PMID: 21975852

 

 

Couthouis J, Hart MP, Erion R, King OD, Diaz Z, Nakaya T, Ibrahim F, Kim HJ, Mojsilovic-Petrovic J, Panossian S, Kim CE, Frackelton EC, Solski JA, Williams KL, Clay-Falcone D, Elman L, McCluskey L, Greene R, Hakonarson H, Kalb RG, Lee VM, Trojanowski JQ, Nicholson GA, Blair IP, Bonini NM, Van Deerlin VM, Mourelatos Z, Shorter J, Gitler AD.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis.

Hum Mol Genet. 2012 Mar 27. [Epub ahead of print]

 

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15, have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo, and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here we report an analysis of EWSR1 in multiple functional assays including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease, and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

 

PMID: 22454397

 

 

Matsumiya LC, Sorge RE, Sotocinal SG, Tabaka JM, Wieskopf JS, Zaloum A, King OD, Mogil JS.

Using the Mouse Grimace Scale to reevaluate the efficacy of postoperative analgesics in laboratory mice.

J Am Assoc Lab Anim Sci. 2012 Jan;51(1):42-9.

 

Postoperative pain management in animals is complicated greatly by the inability to recognize pain. As a result, the choice of analgesics and their doses has been based on extrapolation from greatly differing pain models or the use of measures with unclear relevance to pain. We recently developed the Mouse Grimace Scale (MGS), a facial-expression-based pain coding system adapted directly from scales used in nonverbal human populations. The MGS has shown to be a reliable, highly accurate measure of spontaneous pain of moderate duration, and therefore is particularly useful in the quantification of postoperative pain. In the present study, we quantified the relative intensity and duration of postoperative pain after a sham ventral ovariectomy (laparotomy) in outbred mice. In addition, we compiled dose-response data for 4 commonly used analgesics: buprenorphine, carprofen, ketoprofen, and acetaminophen. We found that postoperative pain in mice, as defined by facial grimacing, lasts for 36 to 48 h, and appears to show relative exacerbation during the early dark (active) photophase. We find that buprenorphine was highly effective in inhibiting postoperative pain-induced facial grimacing in mice at doses equal to or lower than current recommendations, that carprofen and ketoprofen are effective only at doses markedly higher than those currently recommended, and that acetaminophen was ineffective at any dose used. We suggest the revision of practices for postoperative pain management in mice in light of these findings.

 

PMID: 22330867

 

 

Duennwald ML.

Growth assays to assess polyglutamine toxicity in yeast.

J Vis Exp. 2012 Mar 5;(61). pii: 3461.

 

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease (1). Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity (2). At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy's disease (3). The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins (3-8). Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

 

PMID: 22415521

 

 

King OD, Gitler AD, Shorter J.

The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease.

Brain Res. 2012 Jan 21. [Epub ahead of print]

 

Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable 'prion domain' enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimer's disease and Huntington's disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion domains of RNA-binding proteins could underlie the classical non-cell-autonomous emanation of neurodegenerative pathology from originating epicenters to neighboring portions of the nervous system. This article is part of a Special Issue entitled RNA-Binding Proteins.

 

PMID: 22445064

 

Zarringhalam K, Ka M, Kook YH, Terranova J, Suh Y, King OD, Um M 

An open system for automatic home-cage behavioral analysis and its application to male and female mouse models of Huntington's disease.

Behav Brain Res. 2012 Jan 13. [Epub ahead of print]

 

Changes in routine mouse home-cage behavioral activities have been used recently to study alterations of neural circuits caused by genetic and environmental modifications and by drug administration. Nevertheless, automatic assessment of mouse home-cage behaviors remains challenging due to the cost of proprietary systems and to the difficulty in adjusting systems to different monitoring conditions. Here we present software for the automatic quantification of multiple facets of mouse home-cage behaviors, suitable for continuous 24h video monitoring. We used this program to assess behavioral changes in male and female R6/2 transgenic mouse models of Huntington's disease over a 10-week period. Consistent with the well-known progressive motor coordination deficits of R6/2 mice, their hanging, rearing, and climbing activity declined as the disease progressed. R6/2 mice also exhibited frequent disturbances in their resting activity compared to wild-type mice, suggesting that R6/2 mice are more restless and wakeful. Behavioral differences were seen earlier for male R6/2 mice than female R6/2 mice, and "behavioral signatures" based on multiple behaviors enabled us to distinguish male R6/2 mice from sex- and age-matched wild-type controls as early as 5 weeks of age. These results demonstrate that the automated behavioral classification software that we developed ("OpenCage") provides a powerful tool for analyzing natural home-cage mouse behaviors, and for constructing behavioral signatures that will be useful for assessing therapeutic strategies. The OpenCage software is available under an open-source GNU General Public License, allowing other users to freely modify and extend it to suit their purposes.

PMID: 22266926

 

 

Hardt M, Lam DK, Dolan JC, Schmidt BL.

Surveying proteolytic processes in human cancer microenvironments by microdialysis and activity-based mass spectrometry.

Proteomics Clin Appl. 2011 Dec;5(11-12):636-43.

 

Purpose: We present a strategy to survey proteolytic processes that occur in human cancer microenvironments. Experimental design: In situ microdialysis during oral cancer surgery was combined with mass spectrometry-based proteomics to analyze interstitial fluid surrounding tumors and anatomically matched normal sites. Protease activity-based (18)O-profiling was utilized to reveal peptides that were processed by co-collected proteases ex vivo. Results: We demonstrated for the first time the use of microdialysis in humans to collect interstitial fluid from cancer microenvironments. Proteomic profiling identified proteases and inhibitors in the microdialysis samples. A subset of peptides displayed characteristic (18)O-isotope patterns that indicated processing by endogenous proteases. Conclusions and clinical relevance: The presented approach provides unprecedented views of in vivo targets of proteases without disrupting the cancer or surrounding tissue. The methodology can be broadly adapted to other physiological conditions in which proteolytic mediators are involved (e.g. arthritic joints, inflamed muscle, other types of cancer) and where a comparison of normal and pathological tissue is sought.

PMID: 22262628

 

 

Sherley JL.

Overlooked areas need attention for sound evaluation of DNA strand inheritance patterns in Drosophila male germline stem cells

J Cell Sci. 2011 Dec 15;124(Pt 24):4137.

PMID: 22247188

 

 

Klingler D, Hardt M.

Profiling protease activities by dynamic proteomics workflows.

Proteomics. 2012 Jan 13. doi: 10.1002/pmic.201100399. [Epub ahead of print]

 

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.

PMID: 22246865

 

 

Tse JM, Cheng G, Tyrrell JA, Wilcox-Adelman SA, Boucher Y, Jain RK, Munn LL.

Mechanical compression drives cancer cells toward invasive phenotype.

Proc Natl Acad Sci U S A. 2011 Dec 28. [Epub ahead of print]

 

Uncontrolled growth in a confined space generates mechanical compressive stress within tumors, but little is known about how such stress affects tumor cell behavior. Here we show that compressive stress stimulates migration of mammary carcinoma cells. The enhanced migration is accomplished by a subset of "leader cells" that extend filopodia at the leading edge of the cell sheet. Formation of these leader cells is dependent on cell microorganization and is enhanced by compressive stress. Accompanied by fibronectin deposition and stronger cell-matrix adhesion, the transition to leader-cell phenotype results in stabilization of persistent actomyosin-independent cell extensions and coordinated migration. Our results suggest that compressive stress accumulated during tumor growth can enable coordinated migration of cancer cells by stimulating formation of leader cells and enhancing cell-substrate adhesion. This novel mechanism represents a potential target for the prevention of cancer cell migration and invasion.

PMID: 22203958

 

 

Finsen AV, Lunde IG, Sjaastad I, Østli EK, Lyngra M, Jarstadmarken HO, Hasic A, Nygård S, Wilcox-Adelman SA, Goetinck PF, Lyberg T, Skrbic B, Florholmen G, Tønnessen T, Louch WE, Djurovic S, Carlson CR, Christensen G.

Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway.

PLoS One. 2011;6(12):e28302. Epub 2011 Dec 2.

 

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4(-/-) mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4(-/-)-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling.

PMID: 22164265

 

 

Jarousse N, Trujillo DL, Wilcox-Adelman S, Coscoy L.

Virally-induced upregulation of heparan sulfate on B cells via the action of type I IFN.

J Immunol. 2011 Dec 1;187(11):5540-7. Epub 2011 Nov 2.

 

Cell surface heparan sulfate (HS) is an important coreceptor for many cytokines, chemokines, and growth factors. In this study, we report that splenic murine B cells express very little HS and that upon infection with either gammaherpesvirus (murine gammaherpesvirus 68) or betaherpesvirus (murine cytomegalovirus), HS is rapidly upregulated at the surface of B cells. HS upregulation was not observed in mice deficient for the type I IFN (IFN-I) receptor. Additionally, treatment of wild-type mice with the IFN-I inducer polyinosine polycytidylic acid triggered HS expression at the B cell surface. Similarly, incubation of purified splenic B cells with IFN-I, TLR ligands, or BCR stimulators ex vivo resulted in a drastic increase in HS surface expression. We found that IFN-I induced an increase in the surface expression of HS-modified syndecan 4 as well as that of an unidentified heparan sulfate proteoglycan. Finally, IFN-I treatment increased B cell responsiveness to APRIL, a cytokine involved in B cell survival and T cell-independent B cell responses. Enzymatic removal of HS from IFN-I-treated B cells inhibited APRIL. Altogether, our results indicate that upon herpesvirus infection in mice, HS is rapidly upregulated at the surface of B cells due to the action of IFN-I, potentially increasing B cell responsiveness to cytokines. Induction of HS expression at the B cell surface by stimulators of the innate immune response likely plays a key role in the development of a robust immune response.

PMID: 22048770

 

 

Gordon JM, Shikov S, Kuehner JN, Liriano M, Lee E, Stafford W, Poulsen MB, Harrison C, Moore C, Bohm A

Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology.

Biochemistry. 2011 Nov 29;50(47):10203-14. Epub 2011 Nov 2.

 

Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.

PMID: 22026644

 

 

 

Noh M, Smith JL, Huh YH, Sherley JL 

A resource for discovering specific and universal biomarkers for distributed stem cells.

PLoS One. 2011;6(7):e22077.

 

Specific and universal biomarkers for distributed stem cells (DSCs) have been elusive. A major barrier to discovery of such ideal DSC biomarkers is difficulty in obtaining DSCs in sufficient quantity and purity. To solve this problem, we used cell lines genetically engineered for conditional asymmetric self-renewal, the defining DSC property. In gene microarray analyses, we identified 85 genes whose expression is tightly asymmetric self-renewal associated (ASRA). The ASRA gene signature prescribed DSCs to undergo asymmetric self-renewal to a greater extent than committed progenitor cells, embryonic stem cells, or induced pluripotent stem cells. This delineation has several significant implications. These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs. Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification.

PMID: 21818293

 

Jean-François Paré and James L. Sherley

Culture Environment-Induced Pluripotency of SACK-Expanded Tissue Stem Cells 

Journal of Biomedicine and Biotechnology, Volume 2011, Article ID 213457

 

Previous efforts to improve the efficiency of cellular reprogramming for the generation of induced pluripotent stem cells (iPSCs) have focused mainly on transcription factors and small molecule combinations. Here, we report the results of our focus instead on the phenotype of the cells targeted for reprogramming. We find that adult mouse pancreatic tissue stem cells derived by the method of suppression of asymmetric cell kinetics (SACK) acquire increased potency simply by culture under conditions for the production and maintenance of pluripotent stem cells. Moreover, supplementation with the SACK agent xanthine, which promotes symmetric self-renewal, significantly increases the efficiency and degree of acquisition of pluripotency properties. In transplantation analyses, clonal reprogrammed pancreatic stem cells produce slow-growing tumors with tissue derivative of all three embryonic germ layers. This acquisition of pluripotency, without transduction with exogenous transcription factors, supports the concept that tissue stem cells are predisposed to cellular reprogramming, particularly when symmetrically self-renewing.

 

 

Yang Hoon Huh, Johnathan King, Justin Cohen & James L. Sherley 

SACK-Expanded Hair Follicle Stem Cells Display Asymmetric Nuclear Lgr5 Expression With Non-Random Sister Chromatid Segregation 

Sci. Rep. 1, 176  DOI:10.1038/srep00176 (2011)

 

We investigated the properties of clonally-expanded mouse hair follicle stem cells (HF-SCs) in culture. The expansion method, suppression of asymmetric cell kinetics (SACK), is non-toxic and reversible, allowing evaluation of the cells’ asymmetric production of differentiating progeny cells. A tight association was discovered between non-random sister chromatid segregation, a unique property of distributed stem cells (DSCs), like HF-SCs, and a recently described biomarker, Lgr5. We found that nuclear Lgr5 expression was limited to the HF-SC sister of asymmetric self-renewal divisions that retained non-randomly co-segregated chromosomes, which contain the oldest cellular DNA strands, called immortal DNA strands. This pattern-specific Lgr5 association poses a potential highly specific new biomarker for delineation of DSCs. The expanded HF-SCs also maintained the ability to make differentiated hair follicle cells spontaneously, as well as under conditions that induced cell differentiation. In future human cell studies, this capability would improve skin grafts and hair replacement therapies.

 

 

Homma S, Chen JC, Rahimov F, Beermann ML, Hanger K, Bibat GM, Wagner KR, Kunkel LM, Emerson CP Jr, Miller JB 

A unique library of myogenic cells from facioscapulohumeral muscular dystrophy subjects and unaffected relatives: family, disease and cell function.

Eur J Hum Genet. 2011 Nov 23. [Epub ahead of print]

 

To explore possible mechanisms of pathology in facioscapulohumeral muscular dystrophy (FSHD), we generated a novel library of myogenic cells composed of paired cultures derived from FSHD subjects and unaffected first-degree relatives. We prepared cells from biopsies of both biceps and deltoid muscles obtained from each of 10 FSHD and 9 unaffected donors. We used this new collection to determine how family background and disease affected patterns of growth and differentiation, expression of a panel of candidate, and muscle-specific genes, and responses to exogenous stressors. We found that FSHD and unaffected cells had, on average, indistinguishable patterns of differentiation, gene expression, and dose-response curves to staurosporine, paraquat, hydrogen peroxide, and glutathione depletion. Differentiated FSHD and unaffected cultures were both more sensitive to glutathione depletion than proliferating cultures, but showed similar responses to paraquat, staurosporine, and peroxide. For stress responses, the sample size was sufficient to detect a 10% change in effect at the observed variability with a power of >99%. In contrast, for each of these properties, we found significant differences among cells from different cohorts, and these differences were independent of disease status, gender, or muscle biopsied. Thus, though none of the properties we examined could be used to reliably distinguish between FSHD and unaffected cells, family of origin was an important contributor to gene-expression patterns and stressor responses in cultures of both FSHD and unaffected myogenic cells.

PMID: 22108603

 

Li Y, Stafford WF, Hesselberg M, Hayes D, Wu Z, Byrne M.

Characterization of the self-association of human interferon-α2b,albinterferon-α2b, and pegasys.

J Pharm Sci. 2011 Oct 4. doi: 10.1002/jps.22751. [Epub ahead of print]

 

The self-association of human interferon-α2b (hIFN-α2b), albinterferon-α2b (a recombinant protein with human serum albumin and hIFN-α2b peptides fused together in a single polypeptide chain), and Pegasys (PEGylated hIFN-α2a) was

characterized by analytical ultracentrifugation analyses. By examining the apparent sedimentation coefficient distribution profiles of each protein at different concentrations, it was concluded that the above three proteins are self-associating in albinterferon-α2b formulation buffer. By model fitting of sedimentation data using SEDANAL software, the stoichiometry and equilibrium constants of the self-association of these proteins were characterized. The self-association of hIFN-α2b results in the formation of stable dimers, fast-reversible tetramers, octamers, and hexadecamers. In contrast, although both albinterferon-α2b and Pegasys are self-associated, their self-association stoichiometries are significantly different from that of hIFN-α2b. The self-association of albinterferon-α2b results in the formation of reversible dimers and trimers, whereas the self-association of Pegasys gives only reversible dimers. The self-association behaviors of hIFN-α2b and albinterferon-α2b involves attractive electrostatic forces, which can be suppressed to a negligible level in low pH (pH 4.0-4.5) and high salt concentration (400 mM NaCl) buffer, allowing quantification of their size variant contents by sedimentation velocity analysis.

 PMID: 21975852

 

 

Cole JL, Correia JJ, Stafford WF.

The use of analytical sedimentation velocity to extract thermodynamic linkage.

Biophys Chem. 2011 Nov;159(1):120-8. Epub 2011 May 27.

 

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS.

 PMID: 21703752

 

Haithcock J, Billington N, Choi K, Fordham J, Sellers JR, Stafford WF, White H, Forgacs E.

The kinetic mechanism of mouse myosin VIIA.

J Biol Chem. 2011 Mar 18;286(11):8819-28. Epub 2011 Jan 6.

 

Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s(-1) is activated by actin only by ∼3.5-fold resulting in a V(max) of 0.7 s(-1) and a K(ATPase) of 11.5 μM. There is no single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M·T M·D·P) is slow (12 s(-1)) and the equilibrium constant (K(H)) of 1 suggests significant reversal of hydrolysis. In the presence of actin ADP dissociates with a rate constant of 1.2 s(-1). Phosphate dissociation is relatively fast (>12 s(-1)), but the maximal rate could not be experimentally obtained at actin concentrations ≤ 50 μM because of the weak binding of the myosin VIIa-ADP-P(i) complex to actin. At higher actin concentrations the rate of attached hydrolysis (0.4 s(-1)) becomes significant and partially rate-limiting. Our findings suggest that the myosin VIIa is a "slow", monomeric molecular motor with a duty ratio of 0.6.

PMID: 21212272

 

Hayes DB, Stafford WF.

SEDVIEW, real-time sedimentation analysis.

J Biol Chem. 2011 Mar 18;286(11):8819-28. Epub 2011 Jan 6.

 

The ability to obtain a sedimentation coefficient distribution as the runproceeds, and to get an early idea of the quality of a particular sample, has not been made available in real-time during the run in any of the existing software packages. It is desirable on many occasions to be able to see the number of components present in a sample at an early stage of the run. The ability to ascertain the extent of heterogeneity of sample would help enormously to reduce the amount of time that is necessary to obtain that information. Most software packages currently available require that the run be completed before analysis is carried out or at least some of the early scans analyzed off-line to determine if the run should continue. A software package called SEDVIEW has been developed by us to allow early analysis in real-time.

PMID: 20593366

 

Couthouis J, Hart MP, Shorter J, Dejesus-Hernandez M, Erion R, Oristano R, Liu AX, Ramos D, Jethava N, Hosangadi D, Epstein J, Chiang A, Diaz Z, Nakaya T, Ibrahim F, Kim HJ, Solski JA, Williams KL, Mojsilovic-Petrovic J, Ingre C, Boylan K, Graff-Radford NR, Dickson DW, Clay-Falcone D, Elman L, McCluskey L, Greene R, Kalb RG, Lee VM, Trojanowski JQ, Ludolph A, Robberecht W, Andersen PM, Nicholson GA, Blair IP, King OD, Bonini NM, Van Deerlin V, Rademakers R, Mourelatos Z, Gitler AD.

A yeast functional screen predicts new candidate ALS disease genes

Proc Natl Acad Sci U S A. 2011 Nov 7. [Epub ahead of print]

 

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.

PMID: 22065782

 

Duennwald ML.

Polyglutamine misfolding in yeast: Toxic and protective aggregation 

Prion. 2011 Oct 1;5(4). [Epub ahead of print]

 

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.

PMID: 22052348

 

Graceffa P.

Hsp27-actin interaction

Biochem Res Int. 2011;2011:901572

 

Hsp27 oligomer is reported to interact with F-actin as a barbed-end-capping protein. The present study determined the binding strength and stoichiometry of the interaction using fluorescence of probes attached to Hsp27 cysteine-137. The fluorescence of acrylodan attached to Hsp27 increased 4-5-fold upon interaction with F-actin. Titration of the fluorescence with F-actin yielded a weak binding constant (K(D) (app) = 5.3 μM) with an actin/Hsp27 stoichiometry between < 1 and 6. This stoichiometry is inconsistent with an F-actin end-capping protein. Pyrene attached to Hsp27 exhibited a large excimer fluorescence, in agreement with the known proximity of the cysteine-137's in the Hsp27 oligomer. Upon interaction with F-actin the pyrene-Hsp27 excimer fluorescence was largely lost, suggesting that Hsp27 interacts with F-actin as a monomer, consistent with the acrylodan-Hsp27 results. EM images of F-actin-Hsp27 demonstrated that Hsp27 is not a strong G-actin sequester. Thus, Hsp27, in vitro, is a weak F-actin side-binding protein.

PMID: 22007301

 

Lehrer SS.

The 3-state model of muscle regulation revisited: is a fourth state involved?

J Muscle Res Cell Motil. 2011 Nov;32(3):203-8.

 

The 3-state model of muscle regulation has been useful in explaining the roles of Ca(2+) and myosin heads in activation and relaxation of striated muscle contraction. However, there are some phenomena, which cannot simply be explained by the 3-state model. These include increased Ca(2+)-binding caused by strong-binding myosin heads and residual active force at low Ca(2+) in the case of familial hypertrophic cardiomyopathy. Here, I review experimental data which provide evidence for an additional state, a myosin-induced Open state present in the absence of Ca(2+) (Open-Ca(2+)) which like the normal Open+Ca(2+) state, is an active state and can allow myosin heads to cycle and generate force. A schematic diagram is presented which shows that the formation of the Open-Ca(2+) state is on a parallel path with the formation of the Open+Ca(2+) state and can contribute to activation.

PMID: 21948173

 

Jeudy S, Wardrop KE, Alessi A, Dominov JA 

Bcl-2 Inhibits the Innate Immune Response during Early Pathogenesis of Murine Congenital Muscular Dystrophy.

PLoS One. 2011 6(8):e22369. Epub 2011 Aug 5.

 

Laminin α2 (LAMA2)-deficient congenital muscular dystrophy is a severe, early-onset disease caused by abnormal levels of laminin 211 in the basal lamina leading to muscle weakness, transient inflammation, muscle degeneration and impaired mobility. In a Lama2-deficient mouse model for this disease, animal survival is improved by muscle-specific expression of the apoptosis inhibitor Bcl-2, conferred by a MyoD-hBcl-2 transgene. Here we investigated early disease stages in this model to determine initial pathological events and effects of Bcl-2 on their progression. Using quantitative immunohistological and mRNA analyses we show that inflammation occurs very early in Lama2-deficient muscle, some aspects of which are reduced or delayed by the MyoD-hBcl-2 transgene. mRNAs for innate immune response regulators, including multiple Toll-like receptors (TLRs) and the inflammasome component NLRP3, are elevated in diseased muscle compared with age-matched controls expressing Lama2. MyoD-hBcl-2 inhibits induction of TLR4, TLR6, TLR7, TLR8 and TLR9 in Lama2-deficient muscle compared with non-transgenic controls, and leads to reduced infiltration of eosinophils, which are key death effector cells. This congenital disease model provides a new paradigm for investigating cell death mechanisms during early stages of pathogenesis, demonstrating that interactions exist between Bcl-2, a multifunctional regulator of cell survival, and the innate immune response.

PMID: 21850221

 

Huh YH, Sherley JL.

Molecular Cloaking of H2A.Z on Mortal DNA Chromosomes During Non-Random Segregation.

Stem Cells. 2011 10.1002/stem.707. [Epub ahead of print]

 

Although non-random sister chromatid segregation is a singular property of distributed stem cells (DSCs) that are responsible for renewing and repairing mature vertebrate tissues, both its cellular function and its molecular mechanism remain unknown. This situation persists in part because of the lack of facile methods for detecting and quantifying non-random segregating cells and for identifying chromosomes with immortal DNA strands, the cellular molecules that signify non-random segregation. During non-random segregation, at each mitosis, asymmetrically self-renewing DSCs continuously co-segregate to themselves the set of chromosomes that contains immortal DNA strands, which are the oldest DNA strands. Here, we report the discovery of a molecular asymmetry between segregating sets of immortal chromosomes and opposed mortal chromosomes (i.e., containing the younger set of DNA template strands) that constitutes a new convenient biomarker for detection of cells undergoing non-random segregation and direct delineation of chromosomes that bear immortal DNA strands. In both cells engineered with DSC-specific properties and ex vivo-expanded mouse hair follicle stem cells (HF-SCs), the histone H2A variant H2A.Z shows specific immunodetection on immortal DNA chromosomes. Cell fixation analyses indicate that H2A.Z is present on mortal chromosomes as well, but is cloaked from immunodetection; and the cloaking entity is acid-labile. The H2A.Z chromosomal asymmetry produced by molecular cloaking provides a first direct assay for non-random segregation and for chromosomes with immortal DNA strands. It also seems likely to manifest an important aspect of the underlying mechanism(s) responsible for non-random sister chromatid segregation in DSCs.

PMID: 21905168

 

Gangopadhyay JP, Ikemoto N.

Aberrant interaction of calmodulin with the ryanodine receptor develops hypertrophy in the neonatal cardiomyocyte.

Biochem J. 2011 438(2):379-87.

 

We have shown previously that the inter-domain interaction between the two domains of RyR (ryanodine receptor), CaMBD [CaM (calmodulin)-binding domain] and CaMLD (CaM-like domain), activates the Ca2+ channel, and this process is called activation-link formation [Gangopadhyay and Ikemoto (2008) Biochem. J. 411, 415-423]. Thus CaM that is bound to CaMBD is expected to interfere the activation-link formation, thereby stabilizing the closed state of the channel under normal conditions. In the present paper, we report that, upon stimulation of neonatal cardiomyocytes with the pro-hypertrophy agonist ET-1 (endothelin-1), CaM dissociates from the RyR, which induces a series of intracellular events: increased frequency of Ca2+ transients, translocation of the signalling molecules CaM, CaMKII (CaM kinase II) and the transcription factor NFAT (nuclear factor of activated T-cells) to the nucleus. These events then lead to the development of hypertrophy. Importantly, an anti-CaMBD antibody that interferes with activation-link formation prevented all of these intracellular events triggered by ET-1 and prevented the development of hypertrophy. These results indicate that the aberrant formation of the activation link between CaMBD and CaMLD of RyR is a key step in the development of hypertrophy in cultured cardiomyocytes.

PMID: 21649588

 

Mandl A, Huong Pham L, Toth K, Zambetti G, Erhardt P.

Puma deletion delays cardiac dysfunction in murine heart failure models through attenuation of apoptosis.

Circulation. 2011 Jul 5;124(1):31-9. Epub 2011 Jun 13.

 

Background - Puma (p53-upregulated modulator of apoptosis) is a proapoptotic Bcl-2 family protein that serves as a general sensor in response to pathological apoptotic stimuli. In previous work, we demonstrated that puma ablation protects the heart from reperfusion injury in a Langendorff setting. Consistent with this, downregulation of Puma in isolated cardiac myocytes prevented apoptosis induced by different proapoptotic agents. Here, we extended our research to investigate the role of Puma, a downstream mediator of p53, in the development of heart failure using Puma(-/-) mice. Methods and Results - Mice underwent transverse aortic constriction, and the characteristics of cardiac remodeling were analyzed by echocardiography, histology, and gene expression at multiple time points after surgery. Four weeks after the operation, puma deletion attenuated pressure overload-induced apoptosis and fibrosis; however, it did not affect hypertrophy and angiogenesis and maintained functional performance (fractional shortening, 39% versus 25.2% in Puma(-/-) versus WT mice, respectively). Even at 12 weeks after transverse aortic constriction, Puma(-/-) mice displayed only slightly reduced contractility. In addition, transverse aortic constriction induced puma expression in a partially p53-dependent manner. To corroborate these findings, we studied another heart failure model in which heart-specific mdm4 deletion leads to p53 activation and dilated cardiomyopathy. In these mice, Puma was upregulated and its deletion rescued the cardiomyopathy phenotype. Conclusions - Our data indicate that Puma might be a critical component of the apoptotic signaling pathways that contribute to ventricular remodeling and heart failure. Therefore, Puma inactivation may serve as a preferential target to prevent heart failure induced by cellular stress.

PMID: 21670227

 

Kook YH, Ka M, Um M.

Neuroprotective cytokines repress PUMA induction in the 1-methyl-4-phenylpyridinium (MPP(+)) model of Parkinson's disease.

Biochem Biophys Res Commun. 2011 Jun 29. [Epub ahead of print]

 

The hematopoietic cytokines erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) provide neuroprotection in several in vitro and in vivo models of Parkinson's disease (PD). The molecular mechanism by which Epo and G-CSF signals reduce the neuronal death in PD is not clear. Here, we show that in rat pheochromocytoma PC12 cells, Epo and G-CSF efficiently repressed the 1-methyl-4-phenylpyridinium (MPP(+))-induced expression of the proapoptotic protein PUMA (p53 up-regulated modulator of apoptosis). Accordingly, Epo and G-CSF treatment reduced the PC12 cell fraction that underwent apoptosis by MPP(+) treatment and thus improved cell viability. Downregulation of PUMA expression by Epo and G-CSF in MPP(+)-treated PC12 cells seems to be mediated by repression of p53, as the expression of p53 was increased by MPP(+)-treatment and reduced by Epo and G-CSF. Together, these results suggest that the neuroprotective activities of Epo and G-CSF in an experimental model of PD involve the repression of the apoptosis-inducing action of PUMA.

PMID: 21741364

 

Sun CY, van Koningsbruggen S, Long SW, Straasheijm K, Klooster R, Jones TI, Bellini M, Levesque L, Brieher WM, van der Maarel SM, Jones PL.

Facioscapulohumeral Muscular Dystrophy Region Gene 1 Is a Dynamic RNA-Associated and Actin-Bundling Protein.

J Mol Biol. 2011 Jun 15. [Epub ahead of print]

 

FSHD region gene 1 (FRG1) is a dynamic nuclear and cytoplasmic protein that, in skeletal muscle, shows additional localization to the sarcomere. Maintaining appropriate levels of FRG1 protein is critical for muscular and vascular development in vertebrates; however, its precise molecular function is unknown. This study investigates the molecular functions of human FRG1, along with mouse FRG1 and Xenopus frg1, using molecular, biochemical, and cellular-biological approaches, to provide further insight into its roles in vertebrate development. The nuclear fraction of the endogenous FRG1 is localized in nucleoli, Cajal bodies, and actively transcribed chromatin; however, contrary to overexpressed FRG1, the endogenous FRG1 is not associated with nuclear speckles. We characterize the nuclear and nucleolar import of FRG1, the potential effect of phosphorylation, and its interaction with the importin karyopherin α2. Consistent with a role in RNA biogenesis, human FRG1 is associated with mRNA in vivo and invitro, interacts directly with TAP (Tip-associated protein; the major mRNA export receptor), and is a dynamic nuclear-cytoplasmic shuttling protein supporting a function for FRG1 in mRNA transport. Biochemically, we characterize FRG1 actin binding activity and show that the cytoplasmic pool of FRG1 is dependent on an intact actin cytoskeleton for its localization. These data provide the first biochemical activities (actin binding and RNA binding) for human FRG1 and the characterization of the endogenous human FRG1, together indicating that FRG1 is involved in multiple aspects of RNA biogenesis, including mRNA transport and, potentially, cytoplasmic mRNA localization.

PMID: 21699900

 

Bonsor DA, Postel S, Pierce BG, Wang N, Zhu P, Buonpane RA, Weng Z, Kranz DM, Sundberg EJ.

Molecular Basis of a Million-Fold Affinity Maturation Process in a Protein-Protein Interaction.

J Mol Biol. 2011 Jun 12. [Epub ahead of print]

 

Protein engineering is becoming increasingly important for pharmaceutical applications where controlling the specificity and affinity of engineered proteins is required to create targeted protein therapeutics. Affinity increases of several thousand-fold are now routine for a variety of protein engineering approaches, and the structural and energetic bases of affinity maturation have been investigated in a number of such cases. Previously, a 3-million-fold affinity maturation process was achieved in a protein-protein interaction composed of a variant T-cell receptor fragment and a bacterial superantigen. Here, we present the molecular basis of this affinity increase. Using X-ray crystallography, shotgun reversion/replacement scanning mutagenesis, and computational analysis, we describe, in molecular detail, a process by which extrainterfacial regions of a protein complex can be rationally manipulated to significantly improve protein engineering outcomes.

PMID: 21689661

 

Huh YH, Zhou Q, Liao JK, Kitazawa T. 

ROCK inhibition prevents fetal serum-induced alteration in structure and function of organ-cultured mesenteric artery.

J Muscle Res Cell Motil. 2011 Jun 5. [Epub ahead of print]

 

Chronic treatment with fetal bovine serum (FBS) causes contractility reduction, morphological alteration and DNA synthesis in organ-cultured vascular tissues. Here, we tested the hypothesis that chronic inhibition of ROCK has a protective effect on FBS-induced alterations in small arteries. Rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor. Chronic Y-27632 treatment prevented FBS-induced gradual arterial constriction, wall thickening, reduced contractility, and increased ROCK-specific MYPT1 Thr853 phosphorylation. Treatment with Y-27632 also prevented decreased eNOS mRNA expression, and reduced acetylcholine-induced relaxation. Sudden application of Y-27632 to pre-cultured rings reduced MYPT1 phosphorylation and re-widened the constricted rings. Chronic treatment with Y-27632, however, rather augmented than reduced the FBS-induced RhoA over-expression, also increased ROCK1 and MYPT1 expression and averted the FBS-induced reduction of MLC expression, suggesting a compensation of inhibited RhoA/ROCK activity. Sudden removal of Y-27632 caused a rebound in MYPT1 phosphorylation and vasoconstriction in rabbit mesenteric artery. To test which ROCK isoform has greater involvement in FBS-induced contraction, haploinsufficient Rock1 (+/-) and Rock2 (+/-) mouse mesenteric arterial rings were subjected to organ-culture. FBS-induced contraction and RhoA over-expression in either heterozygous animal was not different from wild-type animals. These results suggest that FBS-induced contraction is mediated by up-regulation of RhoA and subsequent activation of ROCK. In conclusion, chronic ROCK inhibition produces some effects that protect against FBS-stimulated vasoconstriction and remodeling. There are also negative effects that a sudden withdrawal of ROCK inhibitor might cause a stronger vasoconstriction than before it was used.

PMID: 21643972

 

 

Fuchs F, Grabarek Z. 

The Ca(2+)/Mg(2+) sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils.

Biochem Biophys Res Commun. 2011 May 20;408(4):697-700.

 

The Ca(2+)/Mg(2+) sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca(2+) was bound to the N-terminal regulatory Ca(2+)-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca(2+). NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1mM or 30μM free Mg(2+). ATPase activity was maximal when sites III and IV were occupied by Mg(2+) and it steadily declined as Ca(2+) displaced Mg(2+). The data suggest that in the absence of Ca(2+) at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg(2+) rather than Ca(2+). This finding could be relevant to the contraction-relaxation kinetics of cardiac muscle. As Ca(2+) dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca(2+) bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.

PMID: 21539814

 

 

Lehrer SS, Ly S, Fuchs F.

Tropomyosin is in a reduced state in rabbit psoas muscle.

J Muscle Res Cell Motil. 2011 May 18. [Epub ahead of print]

 

Tropomyosin (Tm) purified from skeletal and cardiac muscle often contains disulfide bonds due to oxidation of cysteine groups that are in close proximity in the coiled-coil structure. Are these disulfide crosslinks present in the muscle or produced by oxidation during preparation? To answer this question we reacted one part of freshly dissected rabbit psoas muscle fibers, which was permeabilized with Triton X-100, with N-ethyl maleimide (NEM) to block cysteine groups and another part with 5,5'-dithiobis(2-nitro benzoate) (DTNB) to facilitate disulfide bond formation by interchain sulfhydryl-disulfide exchange. We found, by high resolution gradient SDS polyacrylamide gels, that the NEM-treated muscle was only composed of uncrosslinked Tm and the DTNB treated muscle was composed of disulfide-crosslinked Tm. This work indicates that Tm exists in a reduced state in rabbit psoas muscle.

PMID: 21590498

 

 

Homma S, Beermann ML, Miller JB

Peripheral nerve pathology, including aberrant Schwann cell differentiation, is ameliorated by doxycycline in a laminin-{alpha}2-deficient mouse model of congenital muscular dystrophy.

Hum Mol Genet 2011 Apr 19. [Epub ahead of print]

 

The most common form of childhood congenital muscular dystrophy, Type 1A (MDC1A), is caused by mutations in the human LAMA2 gene that encodes the laminin-α2 subunit. In addition to skeletal muscle deficits, MDC1A patients typically show loss of peripheral nerve function. To identify mechanisms underlying this loss of nerve function, we have examined pathology and cell differentiation in sciatic nerves and ventral roots of the laminin-α2-deficient (Lama2(-/-)) mice, which are models for MDC1A. We found that, compared to wild-type, sciatic nerves of Lama2(-/-) mice had a significant increase in both proliferating (Ki67(+)) cells and premyelinating (Oct6(+)) Schwann cells, but also had a significant decrease in both immature/non-myelinating (GFAP(+)) and myelinating (Krox20(+)) Schwann cells. To extend our previous work in which we found that doxycycline, which has multiple effects on mammalian cells, improves motor behavior and more than doubles median life-span of Lama2(-/-) mice, we also determined how nerve pathology was affected by doxycycline treatment. We found that myelinating (Krox20(+)) Schwann cells were significantly increased in doxycycline-treated compared to untreated sciatic nerves. In addition, doxycycline-treated peripheral nerves had significantly less pathology as measured by assays such as amount of unmyelinated or disorganized axons. This study thus identified aberrant proliferation and differentiation of Schwann cells as key components of pathogenesis in peripheral nerves and provided proof-of-concept that pharmaceutical therapy can be of potential benefit for peripheral nerve dysfunction in MDC1A.

PMID: 21505075

 

 

Hishiya A, Salman MN, Carra S, Kampinga HH, Takayama S.

BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity.

PLoS One. 2011 Mar 15;6(3):e16828.

 

A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein αB-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type αB-crystallin and the αB-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by αB-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.

PMID: 21423662

 

Bonsor DA, Sundberg EJ.

Dissecting protein-protein interactions using directed evolution.

Biochemistry. 2011 Apr 5;50(13):2394-402.

 

Protein-protein interactions are essential for life. They are responsible for most cellular functions and when they go awry often lead to disease. Proteins are inherently complex. They are flexible macromolecules whose constituent amino acid components act in combinatorial and networked ways when they engage one another in binding interactions. It is just this complexity that allows them to conduct such a broad array of biological functions. Despite decades of intense study of the molecular basis of protein-protein interactions, key gaps in our understanding remain, hindering our ability to accurately predict the specificities and affinities of their interactions. Until recently, most protein-protein investigations have been probed experimentally at the single-amino acid level, making them, by definition, incapable of capturing the combinatorial nature of, and networked communications between, the numerous residues within and outside of the protein-protein interface. This aspect of protein-protein interactions, however, is emerging as a major driving force for protein affinity and specificity. Understanding a combinatorial process necessarily requires a combinatorial experimental tool. Much like the organisms in which they reside, proteins naturally evolve over time, through a combinatorial process of mutagenesis and selection, to functionally associate. Elucidating the process by which proteins have evolved may be one of the keys to deciphering the molecular rules that govern their interactions with one another. Directed evolution is a technique performed in the laboratory that mimics natural evolution on a tractable time scale that has been utilized widely to engineer proteins with novel capabilities, including altered binding properties. In this review, we discuss directed evolution as an emerging tool for dissecting protein-protein interactions.

PMID: 21332192

 

Fedik Rahimov, Oliver D. King, Leigh C. Warsing, Rachel E. Powell, Charles P. Emerson Jr, Louis M. Kunkel, Kathryn R. Wagner

Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor.

Physiological Genomics 2011, Jan. 25 [Epub ahead of print]

 

Inhibition of the myostatin signaling pathway is emerging as a promising therapeutic means to treat muscle wasting and degenerative disorders. Activin type IIB receptor is the putative myostatin receptor and a soluble activin receptor (ActRIIB-Fc) has been demonstrated to potently inhibit a subset of TGF-ß" family members including myostatin. In order to determine reliable and valid biomarkers for ActRIIB-Fc treatment, we assessed gene expression profiles for quadriceps muscles from mice treated with ActRIIB-Fc compared to mice genetically lacking myostatin and control mice. The expression of 134 genes was found to be significantly altered in mice treated with ActRIIB-Fc over a two-week period relative to control mice (fold-change > 1.5 and P<0.001), whereas the number of significantly altered genes in mice treated for 2 days was 38, demonstrating a time-dependent response to ActRIIB-Fc in overall muscle gene expression. The number of significantly altered genes in Mstn(-/-) mice relative to controls was substantially higher (360), but for most of these genes the expression levels in the two-week treated mice were closer to the levels in the Mstn(-/-) mice than to the control mice (P<10(-30)). Expression levels of 30 selected genes were further validated with qRT-PCR, and a correlation of ≥0.89 was observed between the fold changes from the microarray analysis and the qRT-PCR analysis. These data suggest that treatment with ActRIIB-Fc results in overlapping but distinct gene expression signatures compared to myostatin genetic mutation. Differentially expressed genes identified in this study can be used as potential biomarkers for ActRIIB-Fc treatment, which is currently in clinical trials as a therapeutic agent for muscle wasting and degenerative disorders.

PMID: 21266502 

 

Taghizadeh R, Noh M, Huh YH, Ciusani E, Sigalotti L, Maio M, Arosio B, Nicotra MR, Natali P, Sherley JL

CXCR6, a Newly Defined Biomarker of Tissue-Specific Stem Cell Asymmetric Self-Renewal, Identifies More Aggressive Human Melanoma Cancer Stem Cells.

PLoS One. 2010 Dec 22;5(12):e15183.

 

A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.

We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.

The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

PMID: 21203549

 

Duennwald ML

Monitoring polyglutamine toxicity in yeast.

Methods. 2010 Dec 6. [Epub ahead of print]

 

Experiments in yeast have significantly contributed to our understanding of general aspects of biochemistry, genetics, and cell biology. Yeast models have also delivered deep insights in to the molecular mechanism underpinning human diseases, including neurodegenerative diseases. Many neurodegenerative diseases are associated with the conversion of a protein from a normal and benign conformation into a disease-associated and toxic conformation - a process called protein misfolding. The misfolding of proteins with abnormally expanded polyglutamine (polyQ) regions causes several neurodegenerative diseases, such as Huntington's disease and the Spinocerebellar Ataxias. Yeast cells expressing polyQ expansion proteins recapitulate polyQ length-dependent aggregation and toxicity, which are hallmarks of all polyQ-expansion diseases. The identification of modifiers of polyQ toxicity in yeast revealed molecular mechanisms and cellular pathways that contribute to polyQ toxicity. Notably, several of these findings in yeast were reproduced in other model organisms and in human patients, indicating the validity of the yeast polyQ model. Here, we describe different expression systems for polyQ-expansion proteins in yeast and we outline experimental protocols to reliably and quantitatively monitor polyQ toxicity in yeast.

PMID: 21144902

 

Suzuki Y, Onge RP, Mani R, King OD, Heilbut A, Labunskyy VM, Chen W, Pham L, Zhang LV, Tong AH, Nislow C, Giaever G, Gladyshev VN, Vidal M, Schow P, Lehár J, Roth FP

Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection.

Methods. 2010 Dec 6. [Epub ahead of print]

 

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.

PMID: 21217751

 

Duennwald ML, Shorter J.

Countering amyloid polymorphism and drug resistance with minimal drug cocktails

Prion. 2010 Vol 4, pp.1-8

 

Several fatal, progressive neurodegenerative diseases, including various prion

and prion-like disorders, are connected with the misfolding of specific proteins.

These proteins misfold into toxic oligomeric species and a spectrum of distinct

self-templating amyloid structures, termed strains. Hence, small molecules that

prevent or reverse these protein-misfolding events might have therapeutic

utility. Yet it is unclear whether a single small molecule can antagonize the

complete repertoire of misfolded forms encompassing diverse amyloid polymorphs

and soluble oligomers. We have begun to investigate this issue using the yeast

prion protein Sup35 as an experimental paradigm. We have discovered that a

polyphenol, (-)epigallocatechin-3-gallate (EGCG), effectively inhibited the

formation of infectious amyloid forms (prions) of Sup35 and even remodeled

preassembled prions. Surprisingly, EGCG selectively modulated specific prion

strains and even selected for EGCG-resistant prion strains with novel structural

and biological characteristics. Thus, treatment with a single small molecule

antagonist of amyloidogenesis can select for novel, drug-resistant amyloid

polymorphs. Importantly, combining EGCG with another small molecule,

4,5-bis-(4-methoxyanilino)phthalimide, synergistically antagonized and remodeled

a wide array of Sup35 prion strains without producing any drug-resistant prions.

We suggest that minimal drug cocktails, small collections of drugs that

collectively antagonize all amyloid polymorphs, should be identified to besiege

various neurodegenerative disorders.

PMID: 20935457

 

Jiang SQ, Paulus H.

A high-throughput, homogeneous, fluorescence polarization assay for inhibitors of

hedgehog protein autoprocessing

 J Biomol Screen. 2010 Vol.15, pp.1082-7.

 

Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult

stem cell renewal but has recently been found also to be involved in certain stem

cell cancers. One of the first steps in Hh signaling is the autoprocessing of Hh

protein, in which the C-terminal domain (Hh-C) catalyzes a cholesterol-dependent

autocleavage reaction that leads to the production of the cholesterol ester of

the N-terminal Hh domain (Hh-N), thereby yielding a signaling molecule that

activates the Hh pathway by binding to the Patched receptor. This article

describes an in vitro, homogeneous assay system that measures changes in

fluorescence polarization that accompany the cholesterol-dependent autocleavage

of Hh protein. The assay system makes use of a modified Hh protein in which Hh-N,

which is not essential for autocleavage, is replaced by a 25-residue peptide

containing a tetracysteine motif, complexed with a bisarsenical fluorophore. The

assay is quite robust and easily adapted to high-throughput screening in 384-well

plates with Z' factors above 0.8. It has been used to screen the National

Institutes of Health Clinical Collection, which has led to the identification of

2 compounds that inhibit the cholesterol-dependent autocleavage of Hh protein at

micromolar concentrations.

PMID: 20930213

 

Hishiya A, Kitazawa T, Takayama S.

BAG3 and Hsc70 Interact With Actin Capping Protein CapZ to Maintain Myofibrillar

Integrity Under Mechanical Stress

Circ Res. 2010 Sep 30. [Epub ahead of print]

 

Rationale: A homozygous disruption or genetic mutation of the bag3 gene, a member

of the Bcl-2-associated athanogene (BAG) family proteins, causes cardiomyopathy

and myofibrillar myopathy that is characterized by myofibril and Z-disc

disruption. However, the detailed disease mechanism is not yet fully understood.

Objective: bag3(-/-) mice exhibit differences in the extent of muscle

degeneration between muscle groups with muscles experiencing the most usage

degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests

a role for BAG3 in supporting cytoskeletal connections between the Z-disc and

myofibrils under mechanical stress. The mechanism by which myofibrillar structure

is maintained under mechanical stress remains unclear. The purpose of the study

is to clarify the detailed molecular mechanism of BAG3-mediated muscle

maintenance under mechanical stress. Methods and Results: To address the question

of whether bag3 gene knockdown induces myofibrillar disorganization caused by

mechanical stress, in vitro mechanical stretch experiments using rat neonatal

cardiomyocytes and a short hairpin RNA-mediated gene knockdown system of the bag3

gene were performed. As expected, mechanical stretch rapidly disrupts myofibril

structures in bag3 knockdown cardiomyocytes. BAG3 regulates the structural

stability of F-actin through the actin capping protein, CapZβ1, by promoting

association between Hsc70 and CapZb1. BAG3 facilitates the distribution of CapZβ1

to the proper location, and dysfunction of BAG3 induces CapZ

ubiquitin-proteasome-mediated degradation. Inhibition of CapZβ1 function by

overexpressing CapZβ2 increased myofibril vulnerability and fragmentation under

mechanical stress. On the other hand, overexpression of CapZβ1 inhibits

myofibrillar disruption in bag3 knockdown cells under mechanical stress. As a

result, heart muscle isolated from bag3(-/-) mice exhibited myofibrillar

degeneration and lost contractile activity after caffeine contraction.

Conclusions: These results suggest novel roles for BAG3 and Hsc70 in stabilizing

myofibril structure and inhibiting myofibrillar degeneration in response to

mechanical stress. These proteins are possible targets for further research to

identify therapies for myofibrillar myopathy or other degenerative diseases.

PMID: 20884878

 

Wardrop KE, Dominov JA.

Proinflammatory Signals and the Loss of Lymphatic Vessel Hyaluronan Receptor-1

(LYVE-1) in the Early Pathogenesis of Laminin {alpha}2-Deficient Skeletal Muscle

J Histochem Cytochem. 2010 Sep 27. [Epub ahead of print]

 

Congenital muscular dystrophy type 1A (MDC1A), a severe neuromuscular disease

characterized by early onset muscle weakness and degeneration, is caused by

insufficient levels of laminin α2 (LAMA2) in the basal lamina surrounding muscle

fibers and other cells. A better understanding of the molecular mechanisms

leading to muscle loss is needed in order to develop therapeutic interventions

for this disease. Here we show that inflammation is an early feature of

pathogenesis in Lama2-deficient mouse muscle, indicated by elevated expression of

tenascin C in the endomysium around muscle fibers, infiltration of macrophages,

and induction of the inflammatory cytokines TNFα and IL-1β. In addition, the

expression of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), a

specific marker for lymphatic vessel endothelial cells, is dramatically reduced

early in Lama2-deficient muscle pathogenesis. LYVE-1 expression, which is

inhibited by TNFα, is also decreased in muscles undergoing degeneration due to

dystrophin-deficiency and cardiotoxin damage. LYVE-1 expression thus provides a

useful biomarker to monitor the onset of muscle pathogenesis, likely serving as

an indicator of inflammatory signals present in muscles. Together, our data show

that inflammatory pathways are activated in the earliest stages of

Lama2-deficient disease progression and could play a role in early muscle

degeneration.

PMID: 20876525

 

Wang N, Mattis DM, Sundberg EJ, Schlievert PM, Kranz DM.

A Single, Engineered Protein Therapeutic Neutralizes Exotoxins from Both

Staphylococcus and Streptococcus

 Clin Vaccine Immunol. 2010 Sep 22. [Epub ahead of print] 

 

Staphylococcus aureus and Streptococcus pyogenes secrete exotoxins that act as

superantigens, proteins that cause hyperimmune reactions by binding the variable

domain of the T cell receptor beta chain (Vβ) leading to stimulation of a large

fraction of the T cell repertoire. To develop potential neutralizing agents, we

engineered Vβ mutants with high-affinity for the superantigens staphylococcal

enterotoxin B (SEB), SEC3, and streptococcal pyrogenic exotoxin A (SpeA).

Unexpectedly, the high-affinity Vβ mutants generated against SEB cross-reacted

with SpeA to a greater extent than they did with SEC3, despite greater sequence

similarity between SEB and SEC3. Likewise, the Vβ mutants generated against SpeA

cross-reacted with SEB to a greater extent than with SEC3. The structural basis

of the high-affinity and cross-reactivity was examined by single-site mutational

analyses. The cross-reactivity seems to involve only one or two toxin residues.

Soluble forms of the cross-reactive Vβ regions neutralized both SEB and SpeA in

vivo, suggesting structure-based strategies for generating high-affinity

neutralizing agents that can cross-react with multiple exotoxins.

PMID: 20861327

 

Kitazawa T.

G protein-mediated Ca²+-sensitization of CPI-17 phosphorylation in arterial

smooth muscle

Biochem Biophys Res Commun. 2010 Vol.401 pp.75-8.

 

CPI-17 is a unique phosphoprotein that specifically inhibits myosin light chain

phosphatase in smooth muscle and plays an essential role in agonist-induced

contraction. To elucidate the in situ mechanism for G protein-mediated

Ca²+-sensitization of CPI-17 phosphorylation, α-toxin-permeabilized arterial

smooth muscle strips were used to monitor both force development and CPI-17

phosphorylation in response to GTPγS with varying Ca²+ concentrations. CPI-17

phosphorylation increased at unphysiologically high Ca²+ levels of pCa ≤ 6. GTPγS

markedly enhanced the Ca²+ sensitivity of CPI-17 steady-state phosphorylation but

had no enhancing effect under Ca²+-free conditions, while the potent PKC

activator PDBu increased CPI-17 phosphorylation regardless of Ca²+ concentration.

CPI-17 phosphorylation induced by pCa 4.5 alone was markedly inhibited by the

presence of PKC inhibitor but not ROCK inhibitor. In the presence of calyculin A,

a potent PP1/PP2A phosphatase inhibitor, CPI-17 phosphorylation increased with

time even under Ca²+-free conditions. Furthermore, as Ca²+ concentration

increased, so did CPI-17 phosphorylation rate. GTPγS markedly enhanced the rate

of phosphorylation of CPI-17 at a given Ca²+. In the absence of calyculin A,

either steady-state phosphorylation of CPI-17 under Ca²+-free conditions in the

presence of GTPγS or at pCa 6.7 in the absence of GTPγS was negligible,

suggesting a high intrinsic CPI-17 phosphatase activity. In conclusion,

cooperative increases in Ca²+ and G protein activation are required for a

significant activation of total kinases that phosphorylate CPI-17, which together

overcome CPI-17 phosphatase activity and effectively increase the Ca²+

sensitivity of CPI-17 phosphorylation and smooth muscle contraction.

PMID: 20833141

 

Komaba S, Coluccio LM.

Localization of myosin 1b to actin protrusions requires phosphoinositide binding

 J Biol Chem. 2010 Vol. 285 pp.27686-93.

 

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed,

actin-associated molecular motor. Transient kinetic and single-molecule studies

indicate that it is kinetically slow and responds to tension. Localization and

subcellular fractionation studies indicate that Myo1b associates with the plasma

membrane and certain subcellular organelles such as endosomes and lysosomes.

Whether Myo1b directly associates with membranes is unknown. We demonstrate here

that full-length rat Myo1b binds specifically and with high affinity to

phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatidylinositol

3,4,5-triphosphate (PIP(3)), two phosphoinositides that play important roles in

cell signaling. Binding is not Ca(2+)-dependent and does not involve the

calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the

binding site is contained entirely within the C-terminal tail region, which

contains a putative pleckstrin homology domain. Single mutations in the putative

pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP(2)

and PIP(3) in vitro. These same mutations alter the distribution of Myc-tagged

Myo1b at membrane protrusions in HeLa cells where PIP(2) localizes. In addition,

we found that motor activity is required for Myo1b localization in filopodia.

These results suggest that binding of Myo1b to phosphoinositides plays an

important role in vivo by regulating localization to actin-enriched membrane

projections.

PMID: 20610386

 

Gergely J.

Obituary S.V. Perry

J Muscle Res Cell Motil. 2010 Vol.31 pp.7-8.

PMID: 20512525

 

Wang CL, Coluccio LM.

New insights into the regulation of the actin cytoskeleton by tropomyosin

Int Rev Cell Mol Biol. 2010 Vol.281 pp.91-128.

 

The actin cytoskeleton is regulated by a variety of actin-binding proteins

including those constituting the tropomyosin family. Tropomyosins are coiled-coil

dimers that bind along the length of actin filaments. In muscles, tropomyosin

regulates the interaction of actin-containing thin filaments with

myosin-containing thick filaments to allow contraction. In nonmuscle cells where

multiple tropomyosin isoforms are expressed, tropomyosins participate in a number

of cellular events involving the cytoskeleton. This chapter reviews the current

state of the literature regarding tropomyosin structure and function and

discusses the evidence that tropomyosins play a role in regulating actin

assembly.

PMID: 20460184

 

Gangopadhyay JP, Ikemoto N.

Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein

kinase II during the development of hypertrophy in neonatal cardiomyocytes

Biochem Biophys Res Commun. 2010  Vol.396 pp.515-21

 

We have recently shown that stimulation of cultured neonatal cardiomyocytes with

endothelin-1 (ET-1) first produces conformational disorder within the ryanodine

receptor (RyR2) and diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR),

then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 [3]).

The present paper addresses the following question. By what mechanism does

crosstalk between defective operation of RyR2 and activation of the HT gene

program occur? Here we show that the immuno-stain of calmodulin (CaM) is

localized chiefly in the cytoplasmic area in the control cells; whereas, in the

ET-1-treated/hypertrophied cells, major immuno-staining is localized in the

nuclear region. In addition, fluorescently labeled CaM that has been introduced

into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to

the nucleus with the development of HT. The immuno-confocal imaging of

Ca(2+)/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus

shift of the immuno-staining pattern in the hypertrophied cells. In an early

phase of hypertrophic growth, the frequency of spontaneous Ca(2+) transients

increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a

later phase of hypertrophic growth, further increase in the frequency of

spontaneous Ca(2+) transients results in the appearance of trains of Ca(2+)

spikes, which accompanies with nuclear translocation of CaMKII. The

cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized

inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant

intracellular Ca(2+) events and prevents nuclear translocation of both CaM and

CaMKII, then prevents the development of HT. These results suggest that

translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as

messengers to transmit the pathogenic signal elicited in the surface membrane and

in the RyR2 to the nuclear transcriptional sites to activate HT program.

PMID: 20433809

 

Beermann ML, Ardelt M, Girgenrath M, Miller JB.

Prdm1 (Blimp-1) and the expression of fast and slow myosin heavy chain isoforms

during avian myogenesis in vitro

PLoS One. 2010 Vol.5 p9951.

 

BACKGROUND: Multiple types of fast and slow skeletal muscle fibers form during

early embryogenesis in vertebrates. In zebrafish, formation of the earliest slow

myofibers in fin muscles requires expression of the zinc-finger transcriptional

repressor Prdm1 (also known as Blimp1). To further understand how the role of

Prdm1 in early myogenesis may vary through evolution and during development, we

have now analyzed Prdm1 expression in the diverse types of myotubes that form in

culture from somitic, embryonic, and fetal chicken myoblasts. PRINCIPAL FINDINGS:

In cultures of somitic, embryonic limb, and fetal limb chicken cells, we found

that Prdm1 was expressed in all of the differentiated muscle cells that formed,

including those that expressed only fast myosin heavy chain isoforms, as well as

those that co-expressed both fast and slow myosin heavy chain isoforms. Prdm1 was

also expressed in Pax7-positive myoblasts, as well as in non-myogenic cells in

the cultures. Furthermore, though all differentiated cells in control somite

cultures co-expressed fast and slow myosin heavy chains, antisense knockdown of

Prdm1 expression inhibited the formation of these co-expressing cells in somite

cultures. CONCLUSIONS: In chicken myogenic cell cultures, Prdm1 was expressed in

most Pax7-positive myoblasts and in all differentiated muscle cells, irrespective

of the developmental stage of cell donor or the pattern of fast and slow myosin

heavy chains expressed in the differentiated cells that were formed. Thus, Prdm1

was expressed in myogenic cells prior to terminal differentiation; and, after

differentiation, Prdm1 expression was not limited to cells that expressed slow

myosin heavy chain isoforms. In addition, Prdm1 appeared to be required for

differentiation of the somitic myocytes, which are the earliest myocytes to form

in the avian embryo.

PMID: 20376350

 

Sarkes D, Rameh LE.

A novel HPLC-based approach makes possible the spatial characterization of

cellular PtdIns5P and other phosphoinositides

Biochem J. 2010  Vol.428 pp.375-84.

 

Comment in:  Biochem J. 2010 Jun 15;428(3):e1-2.

 

PtdIns5P was discovered in 1997 [Rameh, Tolias, Duckworth and Cantley (1997)

Nature 390, 192-196], but still very little is known about its regulation and

function. Hitherto, studies of PtdIns5P regulation have been hindered by the

inability to measure cellular PtdIns5P using conventional HPLC, owing to poor

separation from PtdIns4P. In the present paper we describe a new HPLC method for

resolving PtdIns5P from PtdIns4P, which makes possible accurate measurements of

basal and inducible levels of cellular PtdIns5P in the context of other

phosphoinositides. Using this new method, we found that PtdIns5P is

constitutively present in all cells examined (epithelial cells, fibroblasts and

myoblasts, among others) at levels typically 1-2% of PtdIns4P levels. In the

beta-pancreatic cell line BTC6, which is specialized in insulin secretion,

PtdIns5P levels were higher than in most cells (2.5-4% of PtdIns4P). Using

subcellular fractionation, we found that the majority of the basal PtdIns5P is

present in the plasma membrane, but it is also enriched in intracellular membrane

compartments, especially in SER (smooth endoplasmic reticulum) and/or Golgi,

where high levels of PtdIns3P were also detected. Unlike PtdIns3P, PtdIns5P was

also found in fractions containing very-low-density vesicles. Knockdown of PIP4K

(PtdIns5P 4-kinase) leads to accumulation of PtdIns5P in light fractions and

fractions enriched in SER/Golgi, whereas treatment with Brefeldin A results in a

subtle, but reproducible, change in PtdIns5P distribution. These results indicate

that basal PtdIns5P and the PtdIns5P pathway for PtdIns(4,5)P(2) synthesis may

play a role in Golgi-mediated vesicle trafficking.

PMID: 20370717

 

Mabuchi Y, Mabuchi K, Stafford WF, Grabarek Z.

Modular structure of smooth muscle Myosin light chain kinase: hydrodynamic

modeling and functional implications

Biochemistry. 2010 Vol.49 pp.2903-17.

 

Smooth muscle myosin light chain kinase (smMLCK) is a calcium-calmodulin

complex-dependent enzyme that activates contraction of smooth muscle. The

polypeptide chain of rabbit uterine smMLCK (Swiss-Prot entry P29294) contains the

catalytic/regulatory domain, three immunoglobulin-related motifs (Ig), one

fibronectin-related motif (Fn3), a repetitive, proline-rich segment (PEVK), and,

at the N-terminus, a unique F-actin-binding domain. We have evaluated the spatial

arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its

two catalytically active C-terminal fragments (77 kDa, residues 461-1147, and 61

kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to

F-actin show particles at variable distances (11-55 nm) from the filament,

suggesting that a well-structured C-terminal segment of smMLCK is connected to

the actin-binding domain by a long, flexible tether. We have used structural

homology and molecular dynamics methods to construct various all-atom

representation models of smMLCK and its two fragments. The theoretical

sedimentation coefficients computed with HYDROPRO were compared with those

determined by sedimentation velocity. We found agreement between the predicted

and observed sedimentation coefficients for models in which the independently

folded catalytic domain, Fn3, and Ig domains are aligned consecutively on the

long axis of the molecule. The PEVK segment is modeled as an extensible linker

that enables smMLCK to remain bound to F-actin and simultaneously activate the

myosin heads of adjacent myosin filaments at a distance of >or=40 nm. The

structural properties of smMLCK may contribute to the elasticity of smooth muscle

cells.

PMID: 20196616

 

Wohlrab H.

Homodimeric intrinsic membrane proteins. Identification and modulation of

interactions between mitochondrial transporter (carrier) subunits

Biochem Biophys Res Commun. 2010 Vol.393 pp.746-50.

 

Transporter (carrier) proteins of the inner mitochondrial membrane link metabolic

pathways within the matrix and the cytosol with transport/exchange of metabolites

and inorganic ions. Their strict control of these fluxes is required for

oxidative phosphorylation. Understanding the ternary complex transport mechanism

with which most of these transporters function requires an accounting of the

number and interactions of their subunits. The phosphate transporter (PTP, Mir1p)

subunit readily forms homodimers with intersubunit affinities changeable by

mutations. Cys28, likely at the subunit interface, is a site for mutations

yielding transport inhibition or a channel-like transport mode. Such mutations

yield a small increase or decrease in affinity between the subunits. The PTP

inhibitor N-ethylmaleimide decreases subunit affinity by a small amount. PTP

mutations that yield the highest (40%) and the lowest (2%) liposome incorporation

efficiencies (LIE) are clustered near Cys28. Such mutant subunits show the lowest

and highest subunit affinities respectively. The oxaloacetate transporter (Oac1p)

subunit has an almost twofold lower affinity than the PTP subunit. The Oac1p,

dicarboxylate (Dic1p) and PTP transporter subunits form heterodimers with even

lower affinities. These results form a firm basis for detailed studies to

establish the effect of subunit affinities on transport mode and activity and for

the identification of the mechanism that prevents formation of heterodimers that

surely will negatively impact oxidative phosphorylation and ATP levels with

serious consequences for the cell.

PMID: 20171189

 

Huang R, Grabarek Z, Wang CL.

Differential effects of caldesmon on the intermediate conformational states of

polymerizing actin

J Biol Chem. 2010 Vol.285 pp.71-9.

 

The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle

contraction. In non-muscle cells, a shorter CaD isoform co-exists with

microfilaments in the stress fibers at the quiescent state, but the

phosphorylated CaD is found at the leading edge of migrating cells where dynamic

actin filament remodeling occurs. We have studied the effect of a C-terminal

fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by

monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its

phosphorylated form either attenuated or accelerated the pyrene emission

enhancement, depending on whether it was added at the early or the late phase of

actin polymerization. However, the CaD fragment had no effect on the yield of

sedimentable actin, nor did it affect the actin ATPase activity. Our findings can

be explained by a model in which nascent actin filaments undergo a maturation

process that involves at least two intermediate conformational states. If present

at early stages of actin polymerization, CaD stabilizes one of the intermediate

states and blocks the subsequent filament maturation. Addition of CaD at a later

phase accelerates F-actin formation. The fact that CaD is capable of inhibiting

actin filament maturation provides a novel function for CaD and suggests an

active role in the dynamic reorganization of the actin cytoskeleton.

PMID: 19889635