Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

By using DNA nuclease digestion and a quantitative "dual tagging" proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca 2؉ -binding proteins were found in the H2AX complex, the roles of Ca 2؉ were examined. The results indicate that Ca 2؉ /CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.

Introduction Although COVID-19 is transmitted via respiratory droplets, there are multiple gastrointestinal and hepatic manifestations of the disease, including abnormal liver-associated enzymes. However, there are not many published articles on the pathological findings in the liver of COVID-19 patients. Methods We collected the clinical data from 17 autopsy cases of COVID-19 patients including age, sex, BMI, liver function test (ALT, AST, ALP, direct bilirubin and total bilirubin), D-dimer and anticoagulation treatment. We examined histopathologic findings in postmortem hepatic tissue, immunohistochemical (IHC) staining with antibody against COVID spike protein, CD68 and CD61, and electron microscopy. We counted the number of megakaryocytes in liver sections from these COVID-19 positive cases. Results Abnormal liver-associated enzymes were observed in 12/17 cases of COVID-19 infection. With the exception of three cases that had not been tested for D-dimer, all 14 patients’ D-dimer levels were increased, including the cases that received varied doses of anticoagulation treatment. Microscopically, the major findings were widespread platelet-fibrin microthrombi, steatosis, histiocytic hyperplasia in the portal tract, mild lobular inflammation, ischemic-type hepatic necrosis and zone 3 hemorrhage. Rare megakaryocytes were found in sinusoids. COVID IHC demonstrates positive staining of the histiocytes in the portal tract. Under electron microscopy, histiocyte proliferation is present in the portal tract containing lipid droplets, lysosomes, dilated ribosomal endoplasmic reticulum (RER), micro-vesicular bodies and coronavirus. Conclusions The characteristic findings in COVID-19 patients’ liver include numerous amounts of platelet-fibrin microthrombi as well as various degrees of steatosis and histiocytic hyperplasia in the portal tract. Possible mechanisms are also discussed.

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