Biology subjects

Taminishi, S.

Publications and source records attributed to Taminishi, S..

4 recordsLinked to original sources

bioRxiv2023

Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

BackgroundTissue fibrosis is a common feature of many organ dysfunctions, such as heart failure and chronic kidney disease. However, no fundamental treatment has been developed. This study aims to identify novel molecular mechanisms for antifibrotic intervention, focusing on fibroblast activation. MethodsWe performed a forward genetic screen using a genome-wide CRISPR library in the context of transforming growth factor {beta} (TGF-{beta})-mediated connective tissue growth factor (CTGF) expression, and used unbiased techniques such as Cleavage Under Targets and Tagmentation (CUT&Tag) and proximity-dependent biotin labeling by TurboID to reveal the detailed molecular mechanisms. ResultsCRISPR library screening identified a number of players in both the canonical Smad pathway and the non-canonical pathway. In addition to the known factors, the Keap1-Nrf2 pathway was identified as a predominant regulator of TGF-{beta}-mediated CTGF expression. Keap1 deletion and consequent Nrf2 activation broadly suppressed profibrotic gene expression, independently of conventional antioxidant effects. CUT&Tag revealed that Nrf2 bound to the proximity of fibrosis-related genes including Ctgf and Fn1. Subsequent individual analysis revealed Smad3 and RNA polymerase II binding to the Nrf2 peak site, which was attenuated by Keap1 deletion. TurboID experiments further discovered that Nrf2 interacts with Ddx54, which acts as a corepressor. Consistently, Keap1 deletion-mediated repression of profibrotic gene expression was reversed by additional Ddx54 deletion. The impact of the Keap1-Nrf2 pathway on pathological fibrosis was examined using tamoxifen-inducible fibroblast-specific Keap1 knockout mice. Pressure overload for 4 weeks robustly induced cardiac hypertrophy, fibrosis and contractile dysfunction. However, deletion of Keap1 in the Postn lineage attenuated these cardiac pathologies. The anti-fibrotic effects of Keap1 deletion were also confirmed in renal fibrosis in the unilateral ureteral obstruction (UUO) model. ConclusionsFibroblast Nrf2 transcriptionally represses fibrosis-related genes in cooperation with the corepressor Ddx54. Fibroblast-specific deletion of Keap1 attenuated pathological fibrosis in pressure overload heart failure and renal fibrosis.

cell biology↗

bioRxiv2023

Deep mutational scanning of whole SARS-CoV-2 spike in an inverted infection system

In order to investigate SARS-CoV-2 mutations and their impact on immune evasion and infectivity, we developed a Deep Mutational Scanning (DMS) platform utilizing an inverted infection assay to measure spike expression, ACE2 affinity, and viral infectivity in human cells. Surprisingly, our analysis reveals that spike protein expression, rather than ACE2 affinity, is the primary factor affecting viral infectivity and correlated with SARS-CoV-2 evolution. Notably, within the N-terminal domain (NTD), spike expression and infectivity-enhancing mutations are concentrated in flexible loops. We also observed that Omicron variants BA.1 and BA.2 exhibit immune evasion through receptor binding domain (RBD) mutations, although these mutations reduce structural stability. Interestingly, the NTD has evolved to increase stability, compensating for the RBD instability and resulting in heightened overall infectivity. Our findings, available in SpikeScanDB, emphasize the importance of spike expression levels and compensatory mutations in both the NTD and RBD domains for shaping Omicron variant infectivity.

microbiology↗

bioRxiv2022

Liver lipophagy ameliorates nonalcoholic steatohepatitis through lysosomal lipid exocytosis

Nonalcoholic steatohepatitis (NASH) is a progressive disorder with aberrant lipid accumulation and subsequent inflammatory and profibrotic response. Therapeutic efforts at lipid reduction via increasing cytoplasmic lipolysis unfortunately worsens hepatitis due to toxicity of liberated fatty acid. An alternative approach could be lipid reduction through autophagic disposal, i.e., lipophagy. We engineered a synthetic adaptor protein to induce lipophagy, combining a lipid droplet-targeting signal with optimized LC3-interacting domain. Activating hepatocyte lipophagy in vivo strongly mitigated both steatosis and hepatitis in a diet-induced mouse NASH model. Mechanistically, activated lipophagy promoted the excretion of lipid from hepatocytes via lysosomal exocytosis, thereby suppressing harmful intracellular accumulation of nonesterified fatty acid. A high-content compound screen identified alpelisib and digoxin, clinically-approved compounds, as effective activators of lipophagy. Administration of alpelisib or digoxin in vivo strongly inhibited the transition to steatohepatitis. These data thus identify lipophagy as a promising therapeutic approach to prevent NASH progression.

cell biology↗

bioRxiv2021

Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge. One Sentence SummaryOmicron, carrying [~]30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2.

microbiology↗