Graham F. Brady

Regulation of the Copper Chaperone CCS by XIAP-Mediated Ubiquitination

ABSTRACT In order to balance the cellular requirements for copper with its toxic properties, an elegant set of mechanisms has evolved to regulate and buffer intracellular copper. The X-linked inhibitor of apoptosis (XIAP) protein was recently identified as a copper-binding protein and regulator of copper homeostasis, although the mechanism by which XIAP binds copper in the cytosol is unclear. Here we describe the identification of the copper chaperone for superoxide dismutase (CCS) as a mediator of copper delivery to XIAP in cells. We also find that CCS is a target of the E3 ubiquitin ligase activity of XIAP, although interestingly, ubiquitination of CCS by XIAP was found to lead to enhancement of its chaperone activity toward its physiologic target, superoxide dismutase 1, rather than proteasomal degradation. Collectively, our results reveal novel links among apoptosis, copper metabolism, and redox regulation through the XIAP-CCS complex.

Enhanced Cytoprotective Effects of the Inhibitor of Apoptosis Protein Cellular IAP1 through Stabilization with TRAF2

Inhibitor of apoptosis (IAP) proteins are key regulators of intracellular signaling that interact with tumor necrosis factor (TNF) receptor superfamily members as well as proapoptotic molecules such as Smac/DIABLO and caspases. Whereas the X-linked IAP is an established caspase inhibitor, the protective mechanisms utilized by the cellular IAP (c-IAP) proteins are less clear because c-IAPs bind to but do not inhibit the enzymatic activities of caspases. In this study, c-IAPs are shown to be highly unstable molecules that undergo autoubiquitination. The autoubiquitination of c-IAP1 is blocked upon coexpression with TNF receptor-associated factor (TRAF) 2, and this is achieved by inhibition of the E3 ubiquitin ligase activity intrinsic to the RING of c-IAP1. Consistent with these observations, loss of TRAF2 results in a decrease in c-IAP1 levels. Stabilized c-IAP1 was found to sequester and prevent Smac/DIABLO from antagonizing X-linked IAP and protect against cell death. Therefore, this study describes an intriguing cytoprotective mechanism utilized by c-IAP1 and provides critical insight into how IAP proteins function to alter the apoptotic threshold.

Lipogenic transcription factor ChREBP mediates fructose-induced metabolic adaptations to prevent hepatotoxicity

Epidemiologic and animal studies implicate overconsumption of fructose in the development of nonalcoholic fatty liver disease, but the molecular mechanisms underlying fructose-induced chronic liver diseases remain largely unknown. Here, we have presented evidence supporting the essential function of the lipogenic transcription factor carbohydrate response element–binding protein (ChREBP) in mediating adaptive responses to fructose and protecting against fructose-induced hepatotoxicity. In WT mice, a high-fructose diet (HFrD) activated hepatic lipogenesis in a ChREBP-dependent manner; however, in Chrebp-KO mice, a HFrD induced steatohepatitis. In Chrebp-KO mouse livers, a HFrD reduced levels of molecular chaperones and activated the C/EBP homologous protein–dependent (CHOP-dependent) unfolded protein response, whereas administration of a chemical chaperone or Chop shRNA rescued liver injury. Elevated expression levels of cholesterol biosynthesis genes in HFrD-fed Chrebp-KO livers were paralleled by an increased nuclear abundance of sterol regulatory element–binding protein 2 (SREBP2). Atorvastatin-mediated inhibition of hepatic cholesterol biosynthesis or depletion of hepatic Srebp2 reversed fructose-induced liver injury in Chrebp-KO mice. Mechanistically, we determined that ChREBP binds to nuclear SREBP2 to promote its ubiquitination and destabilization in cultured cells. Therefore, our findings demonstrate that ChREBP provides hepatoprotection against a HFrD by preventing overactivation of cholesterol biosynthesis and the subsequent CHOP-mediated, proapoptotic unfolded protein response. Our findings also identified a role for ChREBP in regulating SREBP2-dependent cholesterol metabolism.

A caspase homolog keeps CED-3 in check

Apoptosis is a highly conserved form of cell death that is essential for controlling cell numbers throughout the lifetime of an organism. In Caenorhabditis elegans, the final step in the apoptotic cascade is activation of the death-inducing protease CED-3. Until now, no direct negative regulators of CED-3 had been identified, so the mechanism for maintaining a proper life-death balance was unclear. Now, a new study identifies CSP-3 as an important negative regulator of CED-3 during C. elegans development.

Hepatocyte-Specific Deletion of Mouse Lamin A/C Leads to Male-Selective Steatohepatitis

Background & Aims Lamins are nuclear intermediate filament proteins that comprise the major components of the nuclear lamina. Mutations in LMNA, which encodes lamins A/C, cause laminopathies, including lipodystrophy, cardiomyopathy, and premature aging syndromes. However, the role of lamins in the liver is unknown, and it is unclear whether laminopathy-associated liver disease is caused by primary hepatocyte defects or systemic alterations. Methods To address these questions, we generated mice carrying a hepatocyte-specific deletion of Lmna (knockout [KO] mice) and characterized the KO liver and primary hepatocyte phenotypes by immunoblotting, immunohistochemistry, microarray analysis, quantitative real-time polymerase chain reaction, and Oil Red O and Picrosirius red staining. Results KO hepatocytes manifested abnormal nuclear morphology, and KO mice showed reduced body mass. KO mice developed spontaneous male-selective hepatosteatosis with increased susceptibility to high-fat diet–induced steatohepatitis and fibrosis. The hepatosteatosis was associated with up-regulated transcription of genes encoding lipid transporters, lipid biosynthetic enzymes, lipid droplet-associated proteins, and interferon-regulated genes. Hepatic Lmna deficiency led to enhanced signal transducer and activator of transcription 1 (Stat1) expression and blocked growth hormone–mediated Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), and extracellular signal–regulated kinase (Erk) signaling. Conclusions Lamin A/C acts cell-autonomously to maintain hepatocyte homeostasis and nuclear shape and buffers against male-selective steatohepatitis by positively regulating growth hormone signaling and negatively regulating Stat1 expression. Lamins are potential genetic modifiers for predisposition to steatohepatitis and liver fibrosis. The microarray data can be found in the Gene Expression Omnibus repository (accession number: GSE93643).

Caspases and IAPs: A Dance of Death Ensures Cell Survival

In this issue of Molecular Cell, Ditzel et al. (2008) show that DIAP1 polyubiquitinates DRONC, DCP-1, and drICE, leading to their nondegradative inactivation. Surprisingly, activation of DIAP1 requires caspase-mediated cleavage, revealing an elegant feedback mechanism by which caspases regulate their own fate.

Nuclear lamina genetic variants, including a truncated LAP2, in twins and siblings with nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is becoming the major chronic liver disease in many countries. Its pathogenesis is multifactorial, but twin and familial studies indicate significant heritability, which is not fully explained by currently known genetic susceptibility loci. Notably, mutations in genes encoding nuclear lamina proteins, including lamins, cause lipodystrophy syndromes that include NAFLD. We hypothesized that variants in lamina‐associated proteins predispose to NAFLD and used a candidate gene‐sequencing approach to test for variants in 10 nuclear lamina‐related genes in a cohort of 37 twin and sibling pairs: 21 individuals with and 53 without NAFLD. Twelve heterozygous sequence variants were identified in four lamina‐related genes (ZMPSTE24, TMPO, SREBF1, SREBF2). The majority of NAFLD patients (>90%) had at least one variant compared to <40% of controls (P < 0.0001). When only insertions/deletions and changes in conserved residues were considered, the difference between the groups was similarly striking (>80% versus <25%; P < 0.0001). Presence of a lamina variant segregated with NAFLD independently of the PNPLA3 I148M polymorphism. Several variants were found in TMPO, which encodes the lamina‐associated polypeptide‐2 (LAP2) that has not been associated with liver disease. One of these, a frameshift insertion that generates truncated LAP2, abrogated lamin–LAP2 binding, caused LAP2 mislocalization, altered endogenous lamin distribution, increased lipid droplet accumulation after oleic acid treatment in transfected cells, and led to cytoplasmic association with the ubiquitin‐binding protein p62/SQSTM1. Conclusion: Several variants in nuclear lamina‐related genes were identified in a cohort of twins and siblings with NAFLD; one such variant, which results in a truncated LAP2 protein and a dramatic phenotype in cell culture, represents an association of TMPO/LAP2 variants with NAFLD and underscores the potential importance of the nuclear lamina in NAFLD. (Hepatology 2018;67:1710‐1725).

Lamins and Lamin-associated Proteins in Gastrointestinal Health and Disease

The nuclear lamina is a multi-protein lattice composed of A- and B-type lamins and their associated proteins. This protein lattice associates with heterochromatin and integral inner nuclear membrane proteins, providing links among the genome, nucleoskeleton, and cytoskeleton. In the 1990s, mutations in EMD and LMNA were linked to Emery-Dreifuss muscular dystrophy. Since then, the number of diseases attributed to nuclear lamina defects, including laminopathies and other disorders, has increased to include more than 20 distinct genetic syndromes. Studies of patients and mouse genetic models have pointed to important roles for lamins and their associated proteins in the function of gastrointestinal organs, including liver and pancreas. We review the interactions and functions of the lamina in relation to the nuclear envelope and genome, the ways in which its dysfunction is thought to contribute to human disease, and possible avenues for targeted therapies.

Potential association of LMNA-associated generalized lipodystrophy with juvenile dermatomyositis

BackgroundJuvenile dermatomyositis (JDM) is an auto-immune muscle disease which presents with skin manifestations and muscle weakness. At least 10% of the patients with JDM present with acquired lipodystrophy. Laminopathies are caused by mutations in the lamin genes and cover a wide spectrum of diseases including muscular dystrophies and lipodystrophy. The p.T10I LMNA variant is associated with a phenotype of generalized lipodystrophy that has also been called atypical progeroid syndrome.Case presentationA previously healthy female presented with bilateral proximal lower extremity muscle weakness at age 4. She was diagnosed with JDM based on her clinical presentation, laboratory tests and magnetic resonance imaging (MRI). She had subcutaneous fat loss which started in her extremities and progressed to her whole body. At age 7, she had diabetes, hypertriglyceridemia, low leptin levels and low body fat on dual energy X-ray absorptiometry (DEXA) scan, and was diagnosed with acquired generalized lipodystrophy (AGL). Whole exome sequencing (WES) revealed a heterozygous c.29C > T; p.T10I missense pathogenic variant in LMNA, which encodes lamins A and C. Muscle biopsy confirmed JDM rather than muscular dystrophy, showing perifascicular atrophy and perivascular mononuclear cell infiltration. Immunofluroscence of skin fibroblasts confirmed nuclear atypia and fragmentation.ConclusionsThis is a unique case with p.T10I LMNA variant displaying concurrent JDM and AGL. This co-occurrence raises the intriguing possibility that LMNA, and possibly p.T10I, may have a pathogenic role in not only the occurrence of generalized lipodystrophy, but also juvenile dermatomyositis. Careful phenotypic characterization of additional patients with laminopathies as well as individuals with JDM is warranted.

Imaging in gastric anisakiasis

Gastrointestinal anisakiasis is an uncommon zoonotic parasitic infection caused by consumption of raw or undercooked seafood infected with nematodes of genus Anisakis. Given the non-specific clinical presentation of acute abdomen, nausea, and vomiting these patients are often subject to radiologic imaging. We present ultrasound and computed tomography imaging features in a case of gastric anisakiasis demonstrating characteristic features of diffuse gastric submucosal edema, perigastric stranding and trace ascites that helped to further elaborate the clinical history of uncooked fish consumption prompting timely endoscopic diagnosis and management.