Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging.

BACKGROUND AND AIMS: Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. APPROACH AND RESULTS: Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. CONCLUSIONS: Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.

His74 conservation in the bilin reductase PcyA family reflects an important role in protein-substrate structure and dynamics.

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction of biliverdin IXα's two vinyl groups to produce phycocyanobilin, an essential chromophore for phytochromes, cyanobacteriochromes and phycobiliproteins. Previous site directed mutagenesis studies indicated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits proton transfer. Here, we exploit X-ray crystallography, enzymology and molecular dynamics simulations to understand the functional role of this invariant histidine. The structures of the H74A, H74E and H74Q variants of PcyA reveal that a "conserved" buried water molecule that bridges His74 and catalytically essential His88 is not required for activity. Despite distinct conformations of Glu74 and Gln74 in the H74E and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggesting smaller residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular dynamic simulations further reveal that the crucial active site residue Asp105 is more dynamic in H74A compared to wild-type PcyA and the two other His74 variants, supporting the conclusion that the Ala74 mutation has increased the flexibility of the active site.

Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants.

Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of biliverdin IXalpha to phycocyanobilin--the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semireduced bilin radical intermediates that are profoundly stabilized by selected mutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for this stabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. PCC 6803 PcyA in the initial oxidized and one-electron reduced radical states. Unlike wild-type PcyA, both mutants possess a bilin-interacting axial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radical complex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located for D-ring vinyl reduction. On the basis of these structures and companion (15)N-(1)H long-range HMQC NMR analyses to assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.

A Virtual Crossmatch-based Strategy Facilitates Sharing of Deceased Donor Kidneys for Highly Sensitized Recipients.

BACKGROUND: It is estimated that 19.2% of kidneys exported for candidates with >98% calculated panel reactive antibodies are transplanted into unintended recipients, most commonly due to positive physical crossmatch (PXM). We describe the application of a virtual crossmatch (VXM) that has resulted in a very low rate of transplantation into unintended recipients. METHODS: We performed a retrospective review of kidneys imported to our center to assess the reasons driving late reallocation based on the type of pretransplant crossmatch used for the intended recipient. RESULTS: From December 2014 to October 2017, 254 kidneys were imported based on our assessment of a VXM. Of these, 215 (84.6%) were transplanted without a pretransplant PXM. The remaining 39 (15.4%) recipients required a PXM on admission using a new sample because they did not have an HLA antibody test within the preceding 3 months or because they had a recent blood transfusion. A total of 93% of the imported kidneys were transplanted into intended recipients. There were 18 late reallocations: 9 (3.5%) due to identification of a new recipient medical problem upon admission, 5 (2%) due to suboptimal organ quality on arrival, and only 4 (1.6%) due to a positive PXM or HLA antibody concern. A total of 42% of the recipients of imported kidneys had a 100% calculated panel reactive antibodies. There were no hyperacute rejections and very infrequent acute rejection in the first year suggesting no evidence for immunologic memory response. CONCLUSIONS: Seamless sharing is within reach, even when kidneys are shipped long distances for highly sensitized recipients. Late reallocations can be almost entirely avoided with a strategy that relies heavily on VXM.

Completion of the Vimentin Rod Domain Structure Using Experimental Restraints: A New Tool for Exploring Intermediate Filament Assembly and Mutations.

Electron paramagnetic resonance (EPR) spectroscopy of full-length vimentin and X-ray crystallography of vimentin peptides has provided concordant structural data for nearly the entire central rod domain of the protein. In this report, we use a combination of EPR spectroscopy and molecular modeling to determine the structure and dynamics of the missing region and unite the separate elements into a single structure. Validation of the linker 1-2 (L1-2) modeling approach is demonstrated by the close correlation between EPR and X-ray data in the previously solved regions. Importantly, molecular dynamic (MD) simulation of the constructed model agrees with spin label motion as determined by EPR. Furthermore, MD simulation shows L1-2 heterogeneity, with a concerted switching of states among the dimer chains. These data provide the first ever experimentally driven model of a complete intermediate filament rod domain, providing research tools for further modeling and assembly studies.

Identification of a Novel SYK/c-MYC/MALAT1 Signaling Pathway and Its Potential Therapeutic Value in Ewing Sarcoma.

Purpose: Ewing sarcoma (EWS) is a devastating soft tissue sarcoma affecting predominantly young individuals. Tyrosine kinases (TK) and associated pathways are continuously activated in many malignancies, including EWS; these enzymes provide candidate therapeutic targets.Experimental Design: Two high-throughput screens (a siRNA library and a small-molecule inhibitor library) were performed in EWS cells to establish candidate targets. Spleen tyrosine kinase (SYK) phosphorylation was assessed in EWS patients and cell lines. SYK was inhibited by a variety of genetic and pharmacological approaches, and SYK-regulated pathways were investigated by cDNA microarrays. The transcriptional regulation of MALAT1 was examined by ChIP-qPCR, luciferase reporter, and qRT-PCR assays.Results: SYK was identified as a candidate actionable target through both high-throughput screens. SYK was highly phosphorylated in the majority of EWS cells, and SYK inhibition by a variety of genetic and pharmacologic approaches markedly inhibited EWS cells both in vitro and in vivo Ectopic expression of SYK rescued the cytotoxicity triggered by SYK-depletion associated with the reactivation of both AKT and c-MYC. A long noncoding RNA, MALAT1, was identified to be dependent on SYK-mediated signaling. Moreover, c-MYC, a SYK-promoted gene, bound to the promoter of MALAT1 and transcriptionally activated MALAT1, which further promoted the proliferation of EWS cells.Conclusions: This study identifies a novel signaling involving SYK/c-MYC/MALAT1 as a promising therapeutic target for the treatment of EWS. Clin Cancer Res; 23(15); 4376-87. ©2017 AACR.