A primigravida with very‐long‐chain acyl‐CoA dehydrogenase deficiency

Introduction: Valosin‐containing protein (VCP) is a ubiquitously expressed, multifunctional AAA‐ATPase protein. Its dominant mutations cause hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis. The pattern of muscle weakness in IBMPFD patients is variable and includes limb‐girdle, scapuloperoneal, distal, or axial distributions. Case Report: We report a 63‐year‐old man with progressive scapuloperoneal weakness, head drop, and hyperCKemia since age 40 years. Electromyography showed myopathic changes and rare myotonic discharges. Muscle biopsy revealed numerous lobulated fibers, few fibers with glycogen accumulation, and rare fibers with polyglucosan bodies. Rimmed vacuoles and congophilic inclusions, often seen in IBMPFD, were absent. VCP sequencing identified a novel heterozygous c. 1160G>A mutation resulting in p.Asn387Ser substitution. Conclusions: Our patient broadens the pathological spectrum of VCP‐myopathy and emphasizes the importance of VCP analysis in patients with scapuloperoneal muscular dystrophy despite the absence of Paget disease, dementia, rimmed vacuoles, or intracellular amyloid deposition. Muscle Nerve 50:295–299, 2014     

VCP蛋白表达与胃癌预后的相关性研究

目的：探讨VCP蛋白表达与胃癌预后的关系，确立VCP作为判断胃癌预后的独立因子作用。方法：在187倒胃癌组织手术标本中进行VCP的免疫组织化学染色，比较肿瘤细胞和正常血管内皮细胞VCP的表达，并结合肿瘤浸润、转移、预后等临床因素进行分析比较。结果：VCP的表达与胃癌的浸润、淋巴结转移相关，单变量分析中年龄、胃癌的浸润深度、脉管浸润、淋巴结转移及远处转移等因素显示与胃癌的预后相关（P〈0．05），多变量分析显示了VCP表达水平较肿瘤的分化、淋巴结浸润、转移与胃癌预后相关的危险系数高，故VCP的表达更能反映胃癌的预后。结论：VCP的表达是胃癌重要、独立的预后因子。     

Laryngeal ultrasound detects a high incidence of vocal cord paresis after aortic arch repair in neonates and young children

Objectives

To determine the incidence of vocal cord paresis (VCP) after neonatal aortic arch repair/Norwood-type procedure, and the effectiveness of noninvasive laryngeal ultrasound in detecting VCP compared with gold standard invasive nasoendoscopy.

Jab1 expression is associated with TGF-β1 signaling in chronic rhinosinusitis and nasal polyposis

Jab1, which is a fifth component of COP9 signalosome, plays an essential role in cell growth and proliferation. Jab1 is also shown to regulate transforming growth factor-beta (TGF-β) signaling in carcinoma cells. The aim of the present study was to investigate the expression and the correlation of Jab1 and TGF-β1 in chronic rhinosinusitis and nasal polyposis. Here, we show the elevated expression of Jab1 and TGF-β1 in diseased mucosa without nasal polyps and a correlation between Jab1 and TGF-β1 expression. Forty-six samples (26 patients with nasal polyps, 10 patients with chronic rhinosinusitis and 10 control subjects) were included to this study. Immunohistochemistry and Western blotting were performed for the assessment of Jab1 and TGF-β1 localization and the expression of proteins. Double staining of both proteins showed that Jab1 and TGF-β1 were colocalized in the epithelium, inflammatory cells and the vascular endothelium of nasal mucosa. There was a significant increase in the expression of TGF-β1 and Jab1 in patients without nasal polyps and a significant decrease in patients with nasal polyps compared to controls. Moreover, correlation was detected between the expression of Jab1 and TGF-β1 in chronic rhinosinusitis and nasal polyposis. Our results demonstrate that chronic rhinosinusitis is characterized by elevated expression of Jab1 and TGF-β1 compared to nasal polyposis and Jab1 may play a vital role in the pathogenesis of both chronic rhinosinusitis and nasal polyposis.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Probing allosteric interactions in homo-oligomeric molecular machines using solution NMR spectroscopy

Developments in solution NMR spectroscopy have significantly impacted the biological questions that can now be addressed by this methodology. By means of illustration, we present here a perspective focusing on studies of a number of molecular machines that are critical for cellular homeostasis. The role of NMR in elucidating the structural dynamics of these important molecules is emphasized, focusing specifically on intersubunit allosteric communication in homo-oligomers. In many biophysical studies of oligomers, allostery is inferred by showing that models specifically including intersubunit communication best fit the data of interest. Ideally, however, experimental studies focusing on one subunit of a multisubunit system would be performed as an important complement to the more traditional bulk measurements in which signals from all components are measured simultaneously. Using an approach whereby asymmetric molecules are prepared in concert with NMR experiments focusing on the structural dynamics of individual protomers, we present examples of how intersubunit allostery can be directly observed in high-molecular-weight protein systems. These examples highlight some of the unique roles of solution NMR spectroscopy in studies of complex biomolecules and emphasize the important synergy between NMR and other atomic resolution biophysical methods.

Structural biology, as the name implies, is heavily biased toward the generation of high-resolution biomolecular structures and their interpretation to understand function. Detailed three-dimensional images of proteins have been forthcoming from X-ray crystallography since the late 1950s with the publication of the structure of sperm whale myoglobin by Kendrew et al. (1) and followed subsequently by the structure of human hemoglobin by Muirhead and Perutz in 1963 (2). Since these early seminal achievements, the number of available protein structures has grown exponentially, providing detailed insights into the nature and types of secondary, tertiary, and quaternary structural elements, the mechanisms by which these are stabilized in proteins, and, more generally, how a large variety of different molecular systems might function. A second high-resolution structural method, solution NMR spectroscopy, emerged in the 1980s, largely fueled by a collaboration between Kurt Wüthrich and Richard Ernst. This led, in 1985, to the first three-dimensional solution structure of a protein, the proteinase inhibitor IIA from bull seminal plasma (3). More recently, electron cryomicroscopy (cryo-EM) has emerged as a forefront technology for studies of biomolecular structure (4), and while the focus has been on large molecular complexes, there is great optimism that structures of proteins less than 100 kDa may also become routinely available using this approach (5).With the emergence of cryo-EM, the continued development and application of X-ray methods, and significantly improved predictive algorithms of structure based on artificial intelligence (6, 7), the role of solution NMR spectroscopy as a tool for elucidating detailed structures of proteins has become more ambiguous than in the past. Most certainly, there will still be a need for the experimental determination of atomic resolution structures of relatively small proteins and their complexes in cases in which other structural modalities fail, and the unique ability to investigate molecules in their more natural physiological solution state remains an attractive feature of NMR. Yet both X-ray and cryo-EM methods provide, in general, a more facile and rapid approach for determining structure than NMR spectroscopy. Furthermore, while the motivation behind any X-ray or cryo-EM study is based on the determination of a high-resolution structure (or structures) to inform on some aspect of function as a de facto initial goal, NMR applications can often be, at least initially, less well focused. This has led, over the past several years, to a reevaluation of the role of biomolecular solution NMR spectroscopy in structural biology and biophysics, as its impact as a tool for solving structures has diminished. Yet advances in solution-state NMR studies have been remarkable over the past decades (8 –15), and, now much more than ever before, it is possible to address a wide range of questions focusing on the kinetics, thermodynamics, and dynamics of interactions, both intra- and intermolecular, involving a wide range of biomolecular conformers from folded to intrinsically disordered (16). Insights provided by such studies, pertaining to both biological function and misfunction, are most often simply not available using other technologies.In the perspective that follows, we have chosen to focus on studies of protomer cooperativity and allostery in molecular machines, one area of research in the laboratory, so as to provide examples of how NMR can uniquely shape our understanding of protein science in a manner that is complementary to other biophysical approaches. We have particularly chosen this topic because it is generally not well appreciated that solution NMR spectroscopy can be used to provide quantitative insights into the structural dynamics of large protein complexes with aggregate molecular weights in the many hundreds of kilodaltons. In what follows, the goal is not to be inclusive but to initially focus briefly on the methodology that has led to these applications and then to choose a few examples from systems that we have worked on for the past several years, highlighting unique features that can be obtained through NMR studies.     

Novel p97/VCP inhibitor induces endoplasmic reticulum stress and apoptosis in both bortezomib‐sensitive and ‐resistant multiple myeloma cells

p97/VCP is an endoplasmic reticulum (ER)‐associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER‐associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell‐based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC₅₀, 100‐500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib‐resistant cell line and primary myeloma cells purified from patients. Accumulation of poly‐ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly‐ubiquitinated protein. IC₅₀ of OSSL_325096 to myeloma cell lines were found to be lower (0.1‐0.8 μmol/L) than those of DBeQ (2‐5 μmol/L). In silico protein–drug‐binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell‐free ATPase assays, OSSL_325096 showed dose‐dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti‐myeloma activity, at least in part through p97/VCP inhibition.     

Mitochondrial quality control in amyotrophic lateral sclerosis:towards a common pathway?

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by loss of upper and lower motor neurons. Different mechanisms contribute to the disease initiation and progression, including mitochondrial dysfunction which has been proposed to be a central determinant in ALS patho-genesis. Indeed, while mitochondrial defects have been mainly described in ALS-linked SOD1 mutants, it is now well established that mitochondria become also dysfunctional in other ALS conditions. In such con-text, the mitochondrial quality control system allows to restore normal functioning of mitochondria and to prevent cell death, by both eliminating and replacing damaged mitochondrial components or by degrading the entire organelle through mitophagy. Recent evidence shows that ALS-related genes interfere with the mitochondrial quality control system. This review highlights how ineffective mitochondrial quality control may render motor neurons defenseless towards the accumulating mitochondrial damage in ALS.     

VCP (p97) Regulates NFKB Signaling Pathway, Which Is Important for Metastasis of Osteosarcoma Cell Line

In order to identify genes associated with metastasis, suppression subtractive hybridization (SSH) was performed using murine osteosarcoma cell line Dunn and its subline with higher metastatic potential, LM8. SSH revealed expression of the gene encoding valosin-containing protein (VCP; also known as p97) to be constitutively activated in LM8 cells, but it declined in Dunn cells when the cells became confluent. Because VCP is known to be involved in the ubiquitination process of Inhibitor-αBα (IαBα), an inhibitor of nuclear factor-αB (NFαB), whether VCP influences NFαB activation or not was examined by using VCP-transfected Dunn cells (Dunn/VCPs). When stimulated with tumor necrosis factor-α (TNFα), Dunn/VCPs showed constantly activated NFαB, although in the original Dunn cells and control vector transfectant (Dunn/Dunn-c) NFαB activation ceased when the cells became confluent. Western immunoblot analysis showed an increase of phosphorylated IαBα (p-IKBα) in the cytoplasm of confluent Dunn/Dunn-c cells compared to that of Dunn/VCPs. Therefore, decrease of p-IKBα degrading activity might be responsible for the decrease in NFKB activation. In vitro apoptosis assay demonstrated increased apoptosis rates of Dunn/Dunn-c cells after TNFα stimulation compared to those of Dunn/VCPs and LM8 cells. In vivo metastasis assay showed increased incidences of metastatic events in Dunn/VCP-1 inoculated male C3H mice compared to those in Dunn/Dunn-c inoculated mice. These findings suggested that VCP expression plays an important role in the metastatic process. Anti-apoptotic potential in these cells owing to constant NFKB activation via efficient cytoplasmic p-IKBα degrading activity may explain the increased metastatic potential of these cells.