Mass Spectrometry Analysis of Newly Emerging Coronavirus HCoV-19 Spike Protein and Human ACE2 Reveals Camouflaging Glycans and Unique Post-Translational Modifications

The coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reveal that out of 21 possible glycosites in the HCoV-19 spike protein(S protein),20 are completely occupied by N-glycans,predominantly of the oligomannose type.All seven glycosylation sites in human angiotensin I converting enzyme 2(hACE2)were found to be completely occupied,mainly by complex N-glycans.However,glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2.Additional post-translational modification(PTM)was identified,including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2.Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures.The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19,as well as knowledge for developing desperately needed remedies and vaccines.

Temporal Integrative Omics Reveals an Increase in Nondegradative Ubiquitylation during Primary Hepatocyte Dedifferentiation

Primary hepatocytes(PHCs)are widely used in various fields,but the progressive deterioration of liverspecific features in vitro significantly limits their application.While the transcriptional regulation and whole cell proteome(WCP)of PHCs have been extensively studied,only a small number of studies have addressed the role of posttranslational modifications in this process.To elucidate the underlying mechanisms that induce dedifferentiation,we carried out parallel quantifications of the transcriptome,WCP,ubiquitinome,and phosphoproteome of rat PHCs after 0,6,12,24,and 48 h of in vitro culture.Our data constitute a detailed proteomic analysis of dedifferentiated PHCs including 2196 proteins,2056 ubiquitinated sites,and 4932 phosphorylated peptides.We revealed a low correlation between the transcriptome and WCP during dedifferentiation.A combined analysis of the ubiquitinome with the corresponding WCP indicated that the dedifferentiation of PHCs led to an increase in nondegradative K27 ubiquitination.Functional analysis of the altered phosphoproteins suggested a significant enrichment in ferroptosis.In all,404 proteins with both ubiquitination and phosphorylation were identified to be involved in critical metabolic events.Furthermore,Ptbph Hnqjd,Hnrnpu,and Srrm2 were identified as hub genes.Taken together,our data provide new insights into proteome dynamics during PHC dedifferentiation and potential targets to inhibit the dedifferentiation process.

Human menstrual blood-derived stem cells alleviate autoimmune hepatitis via JNK/MAPK signaling pathway in vivo and in vitro

Autoimmune hepatitis(AIH)is a severe globally distributed liver disease that could occur at any age.Human menstrual blood-derived stem cells(MenSCs)have shown therapeutic effect in acute lung injury and liver failure.However,their role in the curative effect of AIH remains unclear.Here,a classic AIH mouse model was constructed through intravenous injection with concanavalin A(Con A).MenSCs were intravenously injected while Con A injection in the treatment groups.The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated.The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH,mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways.Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation,consistent with the TUNEL staining results.An AML12 co-culture system and JNK inhibitor(SP600125)were used to verify the JNK/MAPK and apoptosis signaling pathways.These findings suggested that MenSCs could be a promising strategy for AIH.

Integrative Proteomic Analysis of Multiple Posttranslational Modifications in Inflammatory Response

Posttranslational modifications(PTMs)of proteins,particularly acetylation,phosphorylation,and ubiquitination,play critical roles in the host innate immune response.PTMs’dynamic changes and the crosstalk among them are complicated.To build a comprehensive dynamic network of inflammation-related proteins,we integrated data from the whole-cell proteome(WCP),acetylome,phosphoproteome,and ubiquitinome of human and mouse macrophages.Our datasets of acetylation,phosphorylation,and ubiquitination sites helped identify PTM crosstalk within and across proteins involved in the inflammatory response.Stimulation of macrophages by lipopolysaccharide(LPS)resulted in both degradative and non-degradative ubiquitination.Moreover,this study contributes to the interpretation of the roles of known inflammatory molecules and the discovery of novel inflammatory proteins.

Integrative proteomics reveals the role of E3 ubiquitin ligase SYVN1 in hepatocellular carcinoma metastasis

Background:Tumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma(HCC),but the relationship between ubiquitination and metastasis need to be studiedmore systematically.We analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC metastasis.Methods:The protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by ubiquitinome.Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligases associated with tumor metastasis.The topological network of protein substrates and corresponding E3 ubiquitin ligaseswas constructed to identify the key E3 ubiquitin ligase.Besides,the growth,migration and invasion ability of LM3 and HUH7 hepatoma cell lines with andwithout SYVN1 expression interferencewere measured by cell proliferation assay,subcutaneous tumor assay,umphal vein endothelium tube formation assay,transwell migration and invasion assays.Finally,the interacting proteins of SYVN1 were screened and verified by protein interaction omics,immunofluorescence,and immunoprecipitation.Ubiquitin levels of related protein substrates in LM3 and HUH7 cells were compared in negative control,SYVN1 knockdown,and SYVN1 overexpression groups.Results:In this study,our whole-cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated sites.We discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression ofHCC metastasis.In addition,proteomic and ubiquitinomic analyses revealed that high expression of E3 ubiquitin-protein ligase SYVN1 is related with tumor metastasis.Furthermore,we found that SYVN1 interacted with heat shock protein 90(HSP90)and impacted the ubiquitination of eukaryotic elongation factor 2 kinase(EEF2K).Conclusions:The ubiquitination profiles of HCC with and without vascular invasion were significantly different.SYVN1 was the most important E3 ubiquitin-protein ligase responsible for this phenomenon,and itwas related with tumormetastasis and growth.Therefore,SYVN1might be a potential therapeutic target for HCC.

Biomimetic hybrid hydrogel for hemostasis,adhesion prevention and promoting regeneration after partial liver resection

Partial liver resection is an established treatment for hepatic disorders.However,surgical bleeding,intra-abdominal adhesion and rapid liver regeneration are still major challenges after partial liver resection,associated with morbidity and mortality.Herein,a biomimetic hybrid hydrogel,composed of oxidized hyaluronic acid,glycol chitosan and MenSCs-derived conditioned medium(CM),is presented to address these issues.The hybrid hydrogel is formed through reversible Schiff base,and possesses injectability and self-healing capability.Moreover,hybrid hydrogel exhibits the capabilities of hemostasis,anti-infection,tissue adhesion and controllable release of cargoes.Based on in vivo studies of the multifunctional hybrid hydrogel,it is demonstrated that acute bleeding in partial liver resection can be ceased immediately by virtue of the hemostasis features of hybrid hydrogel.Also,a significant reduction of intra-abdominal adhesion is confirmed in hybrid hydrogel-treated resection surface.Furthermore,upon the treatment of hybrid hydrogel,hepatic cell proliferation and tissue regeneration can be significantly improved due to the controllably released cytokines from MenSCs-derived CM,exerting the effects of mitogenesis and anti-inflammation in vivo.Thus,the biomimetic hybrid hydrogel can be a promising candidate with great potential for application in partial liver resection.