Shoji Hata

Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1s role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.

Calpain chronicle—an enzyme family under multidisciplinary characterization

Calpain is an intracellular Ca2+-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) discovered in 1964. It was also called CANP (Ca2+-activated neutral protease) as well as CASF, CDP, KAF, etc. until 1990. Calpains are found in almost all eukaryotes and a few bacteria, but not in archaebacteria. Calpains have a limited proteolytic activity, and function to transform or modulate their substrates’ structures and activities; they are therefore called, “modulator proteases.” In the human genome, 15 genes—CAPN1, CAPN2, etc.—encode a calpain-like protease domain. Their products are calpain homologs with divergent structures and various combinations of functional domains, including Ca2+-binding and microtubule-interaction domains. Genetic studies have linked calpain deficiencies to a variety of defects in many different organisms, including lethality, muscular dystrophies, gastropathy, and diabetes. This review of the study of calpains focuses especially on recent findings about their structure–function relationships. These discoveries have been greatly aided by the development of 3D structural studies and genetic models.

A cell-based assay to screen stimulators of the Hippo pathway reveals the inhibitory effect of dobutamine on the YAP-dependent gene transcription

The mammalian Hippo pathway is composed of mammalian Ste20-like (MST) kinases and large tumour suppressor (LATS) kinases. Upon the activation of the pathway, MST kinases phosphorylate and activate LATS kinases, which in turn phosphorylate transcriptional co-activators, yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), recruit them to the cytosol from the nucleus and turn off cell cycle-promoting and anti-apoptotic gene transcriptions. Thus, the pathway restricts cell overgrowth and prevents tumourigenesis. Although a high cell density and stress signallings are known to activate the pathway, no specific stimulators are so far reported. As the dysfunction of the pathway is frequent in human cancers and correlates with poor prognosis, it is important to find out reagents that stimulate the pathway for not only basic research but also clinical medicine. We here developed a cell-based method of screening reagents that induce the recruitment of YAP to the cytosol. Using this method, we found that dobutamine inhibits the YAP-dependent gene transcription. Contrary to our expectations, the effect of dobutamine is independent of the Hippo pathway but our method opens the possibility to discover Hippo pathway stimulators or Hippo-independent YAP inhibitors.

YAP is essential for tissue tension to ensure vertebrate 3D body shape

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D’Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

Dynamic distribution of muscle-specific calpain in mice has a key role in physical-stress adaptation and is impaired in muscular dystrophy

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle-specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretch-induced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.

Neoculin as a New Taste-modifying Protein Occurring in the Fruit of Curculigo latifolia

A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.

Calpain 8/nCL-2 and calpain 9/nCL-4 constitute an active protease complex, G-calpain, involved in gastric mucosal defense.

Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8−/− and Capn9−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8−/− stomach, that of the Capn8CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.

Domain II of m-calpain is a Ca2+-dependent cysteine protease

Calpain, a Ca2+‐dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca2+‐mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m‐calpain, in the absence of Ca2+, revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an ‘open’ conformation, probably due to interactions with other domains. Although the presence of an EF‐hand structure was once predicted in the protease domain, no explicit Ca2+‐binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca2+‐independent protease activity. In this study, we have characterized a truncated human m‐calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca2+‐dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca2+‐dependent modification of the protease domain by the cysteine protease inhibitor, E‐64c, was clearly observed as a SDS–PAGE migration change, indicating that the conformational changes of this domain are a result of Ca2+ binding. These results suggest that the Ca2+ binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.

Liver development and regeneration: from laboratory study to clinical therapy.

The liver has an unusual capacity to regenerate after a loss of mass and function caused by surgical resection or toxic liver injury. Over the last 10 years there have been major advances in our understanding of the molecular and cellular mechanisms underlying liver development and regeneration. The numerous factors crucial to these phenomena have been identified mainly by using knockout mice. Forward‐genetics studies using zebrafish and medaka have also generated many mutants with liver disorders or defects in liver formation. Our goal is to translate knowledge gained from laboratory work and animal models into novel therapies for human liver diseases. Exciting progress has been achieved using human partial liver transplantation and autologous cell therapy.

Possible functions of p94 in connectin-mediated signaling pathways in skeletal muscle cells

Calpains are intracellular Ca2+-requiring ‘modulator proteases’, which modulate cellular functions by limited and specific proteolysis. p94/calpain3, a skeletal-muscle specific calpain, has been one of the representative calpain species which indicates physiological importance of calpain proteolytic system; a defect of proteolytic activity of p94 causes limb girdle muscular dystrophy type2A (LGMD2A, also called ‘calpainopathy’). Immunohistochemical studies on myofibrils showed that p94 localizes at the Z- and N2-line regions of sarcomeres. It was also identified by the yeast two hybrid studies that p94 binds to the N2A and M-line regions of connectin. Furthermore, genetic studies indicate that p94 is indispensable for skeletal muscles, although its precise functions are still unclear. Interestingly, connectin provides sarcomere not only with elasticity but also with binding sites to various multi-functional proteins such as muscle ankyrin repeat proteins (MARPs), muscle RING finger proteins (MURFs), titin-capping protein (T-cap/telethonin), sarcomeric-α-actinin, p94 etc. Binding sites for these proteins are not randomly placed along connectin but rather accumulated in the Z-, N2-, and/or M-line regions, indicating the existence of ‘signal complexes’ unique to each regions. The concept of these complexes are strongly supported by the facts that mutations of connectin or its binding proteins in these regions severely perturb muscle functions, as in the case of LGMD2A caused by mutations in the p94 gene. Therefore, it is hypothesized that the ‘signal complexes’ in the Z-, N2-, and M-lines modulate muscle cell homeostasis by transducing signals of external stimulations/stresses to trigger appropriate response at various different cellular events such as protein modification and gene expressions. In this article, we performed detailed immunohistochemical analyses of p94 on isolated single myofibers. Together with recent findings about p94, it is suggested that sarcomeric localization of p94, especially its M-line localization, is affected by the combination of cellular contexts such as contractile status of myofibrils, fiber type compositions, sarcomeric maturation, and the composition of the ‘signal complexes’ in each region.