Regulatory Myosin Research Articles

The potential mechanisms and regulation of myosin and energy metabolism in endurance training.

Endurance training has great benefits for health and sports performances. However, the genes involved in endurance training and the trigged metabolic activities are still unclear.

Mechanisms of a novel regulatory light chain-dependent cardiac myosin inhibitor.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere. Here, we investigate a novel small molecule, RLC-1, identified in a bovine cardiac myofibril high-throughput screen. RLC-1 is highly dependent on the presence of a regulatory light chain to bind to cardiac myosin and modulate its ATPase activity. In demembranated rat LV trabeculae, RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity of force, while it increased the submaximal rate constant for tension redevelopment. In myofibrils isolated from rat LV, both maximal and submaximal Ca2+-activated force are reduced by nearly 50%. Additionally, the fast and slow phases of relaxation were approximately twice as fast as DMSO controls, and the duration of the slow phase was shorter. Structurally, x-ray diffraction studies showed that RLC-1 moved myosin heads away from the thick filament backbone and decreased the order of myosin heads, which is different from other myosin inhibitors. In intact trabeculae and isolated cardiomyocytes, RLC-1 treatment resulted in decreased peak twitch magnitude and faster activation and relaxation kinetics. In conclusion, RLC-1 accelerated kinetics and decreased force production in the demembranated tissue, intact tissue, and intact whole cells, resulting in a smaller cardiac twitch, which could improve the underlying contractile changes associated with HCM.

CARMIL1 regulates liver cancer cell proliferation by activating the ERK/mTOR pathway through the TRIM27/p53 axis

Capping protein regulatory factor and myosin 1 linker 1 is termed CARMIL1. CARMIL1 is involved in several physiological processes; it forms an actin filament network and plasma membrane-bound cellular projection tissues and positively regulates the cellular components and tissues. CARMIL1 exhibits important biological functions in cancer; nonetheless, these functions have not been completely explored. We aimed to investigate the novel functions of CARMIL1 in liver cancer, particularly in cell proliferation. The cell counting kit-8, 5-ethynyl-2′-deoxyuridine, Component A experiments, and subcutaneous tumor formation model suggest that CARMIL1 is central to the proliferation of liver cancer cells both in vivo and in vitro. We extracted CARMIL1 samples from The Cancer Genome Atlas Program and analyzed its enrichment. CARMIL1 regulated the pathway activity by affecting the expression of star molecular proteins of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Moreover, it influenced the proliferation ability of liver cancer cells. Western blotting suggested that CARMIL1 downregulation could affect ERK and mTOR phosphorylation. Results of the co-immunoprecipitation demonstrated that CARMIL1 binds to tripartite motif (TRIM)27, which in turn binds to p53. Subsequently, CARMIL1 can regulate p53 stability and promote its degradation through TRIM27. Additionally, CARMIL1 inhibition enhanced the sensitivity of liver cancer cells to sorafenib. Tumor growth was significantly inhibited in the group treated with sorafenib and CARMIL1, compared with the group treated with CARMIL1 alone. Sorafenib is a first-line targeted chemotherapeutic drug for hepatocellular carcinoma treatment. It increases the long-term survival of hepatocellular carcinoma by 44%. In this study, downregulated CARMIL1 combined with sorafenib significantly reduced the tumor volume and weight of the mouse subcutaneous tumor model, indicating the potential possibility of combining CARMIL1 with sorafenib in hepatocellular carcinoma treatment. In summary, CARMIL1 promotes liver cancer cell proliferation by regulating the TRIM27/p53 axis and activating the ERK/mTOR pathway.

WNK1 activity modulates biophysical properties of glomerular podocytes through regulation of non-muscle myosin II activation

WNK1 activity modulates biophysical properties of glomerular podocytes through regulation of non-muscle myosin II activation

Smoothelin-like protein 1 promotes insulin sensitivity and modulates the contractile properties of endometrial epithelial cells with insulin resistance.

The incidence of infertility is significantly higher in women with diseases linked to impaired glucose homeostasis, such as insulin resistance. Defective glucose metabolism interferes with fertilization; however, the molecular mechanism underlying this interference is unclear. Smoothelin-like protein 1 (SMTNL1) was isolated from muscle and steroid hormone-responsive tissues and regulates the contractile functions of various cell types through the inhibition of myosin phosphatase (MP) holoenzyme. In addition, SMTNL-1 after phosphorylation at Ser301 by protein kinase A translocates to the nucleus and functions as a transcriptional co-activator of the progesterone receptor-B. SMTNL1 null mice exhibit reduced reproductive fitness and are more prone to type 2 diabetes mellitus. However, the role of SMTNL1 in endometrial epithelial cells is not known. The effect of SMTNL1 overexpression was investigated in pregnancy and in gestational diabetic endometrial epithelial cell models by immunofluorescent staining, cell migration, and semi quantitative Western blot analysis and glucose uptake assay. We show that SMTNL1 promotes the differentiation of endometrial epithelial cells in a progesterone-dependent manner to attenuate insulin resistance. Furthermore, SMTNL1 hampers the migration capacity of epithelial cells in a gestational diabetes model by inhibiting the expression of MYPT1, the regulatory subunit of MP, and the activity of the holoenzyme, resulting in increased phosphorylation of the 20 kDa regulatory myosin light chain. SMTNL1 also acts as an insulin-sensitizing agent by increasing the gene expression of PP2A and DUPS9 protein phosphatases, resulting in decreased ERK1/2 activity and, hence, decreasing the phosphorylation of IRS-1 at Ser612 under gestational diabetes conditions. SMTNL1 may have therapeutic relevance to the progesterone-dependent inhibition of endometrial epithelial cell migration under hyperglycemic conditions and insulin sensitivity in the endometrium in gestational diabetes or other metabolic disorders.

Rhotekin regulates axon regeneration through the talin-Vinculin-Vinexin axis in Caenorhabditis elegans.

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.

Long-term experimental hyperglycemia does not impair macrovascular endothelial barrier integrity and function in vitro

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

Myosin light chain dephosphorylation by ppp1r12c promotes atrial hypocontractility in atrial fibrillation

Abstract Background Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls (P=0.02, n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation (P=0.000001, n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=0.03 and 0.007 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls (P=5.0x10-6, n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls (P=0.02 and 0.04 respectively, n= 6,6,5). Conclusions AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key regulator of atrial contractility in AF.

Myosin and tropomyosin-troponin complementarily regulate thermal activation of muscles.

Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy. Rapid heating (25 to 41.5°C) within 2 s induced reversible sarcomere shortening in relaxing solution. Further, we investigated the temperature-dependence of the sliding velocity of reconstituted fast skeletal or cardiac thin filaments on fast skeletal or β-cardiac myosin in an in vitro motility assay within the body temperature range. We found that (a) with fast skeletal thin filaments on fast skeletal myosin, the temperature dependence was comparable to that obtained for sarcomere shortening in fast skeletal myofibrils (Q10 ∼8), (b) both types of thin filaments started to slide at lower temperatures on fast skeletal myosin than on β-cardiac myosin, and (c) cardiac thin filaments slid at lower temperatures compared with fast skeletal thin filaments on either type of myosin. Therefore, the mammalian striated muscle may be fine-tuned to contract efficiently via complementary regulation of myosin and tropomyosin-troponin within the body temperature range, depending on the physiological demands of various circumstances.

How myosin VI traps its off-state, is activated and dimerizes

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.

Potentiation of active force by cyclic strain in sheep carotid arterial smooth muscle.

The ability to generate force in large arteries is known to be augmented by cyclic strain that mimics the mechanically dynamic in vivo environment associated with blood pressure fluctuation experienced by these arteries. Cyclic strain does not induce a contractile response, like that observed in the myogenic response seen in small arteries, but prompts a substantial increase in the response to electrical stimulation. We coined this phenomenon "force potentiation." Because protein kinase C (PKC) and rho-kinase (ROCK) are known to play a role in increasing contractility of arterial smooth muscle by inhibition of myosin light chain phosphatase, and integrin-link kinase (ILK) is crucial in mechanotransduction, we examined how inhibition of these kinases affected force potentiation in sheep carotid artery. We found that phosphorylation of the regulatory myosin light chain was enhanced by cyclic strain, but the enhancement was observed only in activated, not in relaxed muscle. Inhibition of ROCK diminished force potentiation and active isometric force, likely due to the disinhibition of myosin light chain phosphatase. Inhibition of PKC abolished force potentiation without an effect on active force, suggesting a more exclusive role of PKC (compared with ROCK) in mediating force potentiation. Inhibition of ILK had a similar effect as PKC inhibition, suggesting that ILK may be an upstream kinase for PKC activation by mechanical stimuli. Taken together, the findings suggest that ILK, PKC, and ROCK are important kinases in the signal transduction pathway that mediate the effect of mechanical strain on force potentiation.NEW & NOTEWORTHY When subjected to mechanical strain, smooth muscle from large arteries has the ability to increase its force generation (force potentiation), which could be important in autoregulation of blood pressure. This phenomenon, however, does not involve a myogenic response, such as the one seen in small arteries and arterioles. Our work shows the involvement of ILK, PKC, and ROCK in the signal transduction pathway that mediates the force-potentiating effect of mechanical strain in large arteries.

Divergence of cytokinesis and dimorphism control by myosin II regulatory light chain in fission yeasts

Non-muscle myosin II activation by regulatory light chain (Rlc1Sp) phosphorylation at Ser35 is crucial for cytokinesis during respiration in the fission yeast Schizosaccharomyces pombe. We show that in the early divergent and dimorphic fission yeast S.japonicus non-phosphorylated Rlc1Sj regulates the activity of Myo2Sj and Myp2Sj heavy chains during cytokinesis. Intriguingly, Rlc1Sj-Myo2Sj nodes delay yeast to hyphae onset but are essential for mycelial development. Structure-function analysis revealed that phosphorylation-induced folding of Rlc1Sp α1 helix into an open conformation allows precise regulation of Myo2Sp during cytokinesis. Consistently, inclusion of bulky tryptophan residues in the adjacent α5 helix triggered Rlc1Sp shift and supported cytokinesis in absence of Ser35 phosphorylation. Remarkably, unphosphorylated Rlc1Sj lacking the α1 helix was competent to regulate S.pombe cytokinesis during respiration. Hence, early diversification resulted in two efficient phosphorylation-independent and -dependent modes of Rlc1 regulation of myosin II activity in fission yeasts, the latter being conserved through evolution.

Abstract EC.02: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atria Hypocontractility In Atrial Fibrillation

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls with > 40% reduction in MLC2a phosphorylation. 12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls, with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key regulator of atrial contractility in AF.

Long-Term Experimental Hyperglycemia Does Not Impair Macrovascular Endothelial Barrier Integrity and Function in vitro.

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

Myosin and upalpha-actinin regulation of stress fiber contractility under tensile stress

Stress fibers are actomyosin bundles that regulate cellular mechanosensation and force transduction. Interacting with the extracellular matrix through focal adhesion complexes, stress fibers are highly dynamic structures regulated by myosin motors and crosslinking proteins. Under external mechanical stimuli such as tensile forces, the stress fiber remodels its architecture to adapt to external cues, displaying properties of viscoelastic materials. How the structural remodeling of stress fibers is related to the generation of contractile force is not well understood. In this work, we simulate mechanochemical dynamics and force generation of stress fibers using the molecular simulation platform MEDYAN. We model stress fiber as two connecting bipolar bundles attached at the ends to focal adhesion complexes. The simulated stress fibers generate contractile force that is regulated by myosin motors and alpha-actinin crosslinkers. We find that stress fibers enhance contractility by reducing the distance between actin filaments to increase crosslinker binding, and this structural remodeling ability depends on the crosslinker turnover rate. Under tensile pulling force, the stress fiber shows an instantaneous increase of the contractile forces followed by a slow relaxation into a new steady state. While the new steady state contractility after pulling depends only on the overlap between actin bundles, the short-term contractility enhancement is sensitive to the tensile pulling distance. We further show that this mechanical response is also sensitive to the crosslinker turnover rate. Our results provide new insights into the stress fiber mechanics that have significant implications for understanding cellular adaptation to mechanical signaling.

Abstract 4248: Epithelial ovarian cancer causes cardiac dysfunction and myofilament dysregulation

Abstract Introduction: The intersections between cancer and cardiovascular disease (CVD) have become increasingly relevant in the care of both patient populations. To date, the cardio-oncology field has largely focused on how cancer treatment influences long-term cardiac health. Indeed, many cancer therapeutics show various toxic effects to the cardiovascular system. However, studies have also shown cardiac deficits in therapy-naïve cancer patients. This suggests that these two disease states interact through similar signaling pathways, yet the mechanisms underlying cancer-induced cardiac dysfunction remain unknown. Thus, we investigated cardiac structure and function throughout the progression of epithelial ovarian cancer (EOC). Methods: We used an orthotopic, syngeneic mouse model of ovarian cancer. Briefly, transformed murine ovarian surface epithelial cells from C57BL/6 mice (ID8; 1.0 × 106), or saline for surgical controls, were injected under the ovarian bursa of syngeneic mice. In this model, 60 days after tumor induction, mice form large ovarian masses, numerous peritoneal lesions, and develop abdominal ascites - consistent with the clinical features of stage III (advanced) EOC. To determine whether cardiac abnormalities precede the presence of advanced disease, at 45 days post-tumor induction, mice were anesthetized, and left ventricular (LV) structure and function were assessed by echocardiography and invasive hemodynamics, respectively. Myofilament activity was measured with an actomyosin MgATPase assay and protein phosphorylation was determined by SDS-PAGE with Pro-Q Diamond phosphoprotein staining. Results: EOC mice showed systolic (i.e., reduced contraction rate) and diastolic dysfunction (i.e., reduced relaxation rate, elevated diastolic filling pressure), with no evidence of hypertrophy or atrophy (i.e., no change in LV wall thickness or dimension). While there were no observed differences in myofilament ATPase activity, phosphorylation of regulatory myosin light chain 2 (MYL2) and cardiac myosin binding protein C (cMyBP-C) decreased, and tropomyosin (Tpm) phosphorylation increased in EOC mice. Conclusion: Together, these data demonstrate that ovarian cancer impairs cardiac function and alters the regulation of myofilament proteins critical for contraction and relaxation. Interestingly, the observed dephosphorylation of MYL2 and cMyBP-C and hyperphosphorylation of Tpm are consistent with features of heart failure. The findings build upon our current understanding of the interactions between cancer and CVD and show that deficits in the heart precede the development of advanced malignancy. Future work further will investigate the tumor-heart axis to identify how tumor development disrupts normal cardiac physiology. Citation Format: Leslie M. Ogilvie, Madison Pereira, Kathy Matuszewska, Bridget Coyle-Asbil, Luca J. Delfinis, Shivam Gandhi, Keith R. Brunt, Christopher G. Perry, W. Glen Pyle, Jim Petrik, Jeremy A. Simpson. Epithelial ovarian cancer causes cardiac dysfunction and myofilament dysregulation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4248.

Opportunities and Challenges for the Development of MRCK Kinases Inhibitors as Potential Cancer Chemotherapeutics.

Cytoskeleton organization and dynamics are rapidly regulated by post-translational modifications of key target proteins. Acting downstream of the Cdc42 GTPase, the myotonic dystrophy-related Cdc42-binding kinases MRCKα, MRCKβ, and MRCKγ have recently emerged as important players in cytoskeleton regulation through the phosphorylation of proteins such as the regulatory myosin light chain proteins. Compared with the closely related Rho-associated coiled-coil kinases 1 and 2 (ROCK1 and ROCK2), the contributions of the MRCK kinases are less well characterized, one reason for this being that the discovery of potent and selective MRCK pharmacological inhibitors occurred many years after the discovery of ROCK inhibitors. The disclosure of inhibitors, such as BDP5290 and BDP9066, that have marked selectivity for MRCK over ROCK, as well as the dual ROCK + MRCK inhibitor DJ4, has expanded the repertoire of chemical biology tools to study MRCK function in normal and pathological conditions. Recent research has used these novel inhibitors to establish the role of MRCK signalling in epithelial polarization, phagocytosis, cytoskeleton organization, cell motility, and cancer cell invasiveness. Furthermore, pharmacological MRCK inhibition has been shown to elicit therapeutically beneficial effects in cell-based and in vivo studies of glioma, skin, and ovarian cancers.

Beyond Chaperoning: UCS Proteins Emerge as Regulators of Myosin-Mediated Cellular Processes.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.

Diabetes-induced chronic heart failure is due to defects in calcium transporting and regulatory contractile proteins: cellular and molecular evidence.

Heart failure (HF) is a major deteriorating disease of the myocardium due to weak myocardial muscles. As such, the heart is unable to pump blood efficiently around the body to meet its constant demand. HF is a major global health problem with more than 7 million deaths annually worldwide, with some patients dying suddenly due to sudden cardiac death (SCD). There are several risk factors which are associated with HF and SCD which can negatively affect the heart synergistically. One major risk factor is diabetes mellitus (DM) which can cause an elevation in blood glucose level or hyperglycaemia (HG) which, in turn, has an insulting effect on the myocardium. This review attempted to explain the subcellular, cellular and molecular mechanisms and to a lesser extent, the genetic factors associated with the development of diabetes- induced cardiomyopathy due to the HG which can subsequently lead to chronic heart failure (CHF) and SCD. The study first explained the structure and function of the myocardium and then focussed mainly on the excitation-contraction coupling (ECC) processes highlighting the defects of calcium transporting (SERCA, NCX, RyR and connexin) and contractile regulatory (myosin, actin, titin and troponin) proteins. The study also highlighted new therapies and those under development, as well as preventative strategies to either treat or prevent diabetic cardiomyopathy (DCM). It is postulated that prevention is better than cure.

Cofilin-1 and profilin-1 expression in lung microvascular endothelial cells exposed to titanium dioxide nanoparticles

Air pollutants exacerbate chronic airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). However, the underlying mechanisms are yet to be determined. While a number of studies have reported adverse effects of nanoparticles on humans, little is known about their effects on the respiratory system. To examine the protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to titanium dioxide (TiO2) nanoparticles, a common air pollutant. A proteomics approach using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was used to determine the differences in protein expression at 8 h and 24 h, following the treatment of HMVEC-L with 20-μM or 40-μM TiO2 nanoparticles. Human lung microvascular endothelial cells treated with 20-μM TiO2 nanoparticles showed alterations of 7 protein spots, including molecules related to calcium regulation, transport, cytoskeleton, and muscle contraction. The treatment of HMVEC-L with 40-μM TiO2 nanoparticles resulted in alterations of 4 protein spots, with molecular functions related to the cytoskeleton, myosin regulation, actin modulation, as well as guanosine diphosphate (GDP) and guanosine triphosphate (GTP) regulation. To validate these results, immunohistochemical staining and western blotting analyses were performed on lung tissues collected from mice exposed to TiO2 nanoparticles. Cofilin-1 and profilin-1 were expressed in the endothelium, epithelium and inflammatory cells, and decreased in lung tissues of TiO2 nanoparticle-exposed mice compared to sham-treated controls. These results suggest that some of the differentially expressed proteins may play important roles in airway diseases caused by TiO2 nanoparticle exposure.