Pathologic Proteolytic Processing of N-Cadherin as a Marker of Human Fibrotic Disease

Prior research has implicated the involvement of cell adhesion molecule N-cadherin in tissue fibrosis and remodeling. We hypothesize that anomalies in N-cadherin protein processing are involved in pathological fibrosis. Diseased tissues associated with fibrosis of the heart, lung, and liver were probed for the precursor form of N-cadherin, pro-N-cadherin (PNC), by immunohistochemistry and compared to healthy tissues. Myofibroblast cell lines were analyzed for cell surface pro-N-cadherin by flow cytometry and immunofluorescent microscopy. Soluble PNC products were immunoprecipitated from patient plasmas and an enzyme-linked immunoassay was developed for quantification. All fibrotic tissues examined show aberrant PNC localization. Cell surface PNC is expressed in myofibroblast cell lines isolated from cardiomyopathy and idiopathic pulmonary fibrosis but not on myofibroblasts isolated from healthy tissues. PNC is elevated in the plasma of patients with cardiomyopathy (p ≤ 0.0001), idiopathic pulmonary fibrosis (p ≤ 0.05), and nonalcoholic fatty liver disease with cirrhosis (p ≤ 0.05). Finally, we have humanized a murine antibody and demonstrate that it significantly inhibits migration of PNC expressing myofibroblasts. Collectively, the aberrant localization of PNC is observed in all fibrotic tissues examined in our study and our data suggest a role for cell surface PNC in the pathogenesis of fibrosis.

YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity

The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity.

Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease. Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, will inform our understanding of this disease and its treatment.

Glycosylation of ALV-J envelope protein at sites 17 and 193 is pivotal in the virus replication

Glycans on envelope glycoprotein (Env) of the subgroup J avian leukosis virus (ALV-J) play an essential role in virion integrity and infection process. In this study, we found that among the 13 predicted N-linked glycosylation sites (NGSs) in gp85 of Tibetan chicken strain TBC-J6, N17 and N193/N191 are pivotal in the virus replication. Further research illustrated that mutation at N193 weakened Env-receptor binding in blocking assay of viral entrance, co-immunoprecipitation and ELISA. Our studies also showed that N17 was involved in Env protein processing and later virion incorporation, based on the detection of p27 and Env protein in the supernatant and gp37 in the cell culture. This report is a systematic research on clarifying the biological function of NGSs on ALV-J gp85 , which would provide valuable insights in the role of gp85 in ALV life cycle as well as anti-ALV-J strategies. Importance ALV-J is a retrovirus that can cause multiple types of tumors in chickens. Among all the viral proteins, the heavily glycosylated envelope protein is especially crucial. Glycosylation plays a major role in Env protein function, including protein processing, receptor attachment and immune evasion. Notably, viruses isolated recently seem to lose the 6 th and 11 st NGSs, which are proved to be important in receptor binding. In our study, the 1 st (N17) and 8 th (N193) NGS of gp85 of strain TBC-J6 can largely influence the titer of this virus. Deglycosylation at N193 weakened Env-receptor binding, while mutation at N17 influenced Env protein processing. This study systemically analyzed the function of NGSs in ALV-J in different aspects, which may help us to understand the lifecycle of ALV-J and provide antiviral targets for the control of ALV-J.

Identification of biomarkers and signaling pathways during the treatment of multiple myeloma

Multiple myeloma (MM) is an incurable hematologic malignancy, which is characterized by increased bone marrow plasma cells, osteolytic lesions, and anemia. Here, our aim is to characterize significant biomarkers and signaling pathways during the treatment of MM by using bioinformatic methods. The GSE180018 dataset was generated by DNBSEQ-G400 (Homo sapiens). The KEGG and GO analyses showed the biological processes such as " Protein export”, “Protein processing in endoplasmic reticulum”, “Vibrio cholerae infection”, “Fc gamma R-mediated phagocytosis" are mainly affected in FOXM1 KO MM cells. Moreover, we identified the significant genes including CD44, NOTCH1, SELL, RAC2, HSPA8, VAV1, DDIT3, PLK1, HYOU1, ITGAL in FOXM1 KO MM cells. Therefore, our study may provide further guidance for the drug development of MM.

Cellular Processes

Although cells in the nervous system contain all the cellular machinery that exists in other cells throughout the body, nervous system cells have many specialized functions that present unique challenges in the maintenance of cellular functionality and homeostasis. The unique morphology of neurons demands elaborate cytoarchitecture, energy production, and cellular processing machinery that are unparalleled in other parts of the body. This chapter presents a broad overview of cellular processes related to the cytoskeleton, axonal transport, protein processing, and energy metabolism within the nervous system. Breakdowns of these intricate processes are involved in many diseases of the nervous system, which are discussed in subsequent chapters.

Extracellular calcium alters calcium-sensing receptor network integrating intracellular calcium-signaling and related key pathway

G-protein-coupled receptors (GPCRs) are a target for over 34% of current drugs. The calcium-sensing receptor (CaSR), a family C GPCR, regulates systemic calcium (Ca2+) homeostasis that is critical for many physiological, calciotropical, and noncalciotropical outcomes in multiple organs. However, the mechanisms by which extracellular Ca2+ (Ca2+ex) and the CaSR mediate networks of intracellular Ca2+-signaling and players involved throughout the life cycle of CaSR are largely unknown. Here we report the first CaSR protein–protein interactome with 94 novel putative and 8 previously published interactors using proteomics. Ca2+ex promotes enrichment of 66% of the identified CaSR interactors, pertaining to Ca2+ dynamics, endocytosis, degradation, trafficking, and primarily to protein processing in the endoplasmic reticulum (ER). These enhanced ER-related processes are governed by Ca2+ex-activated CaSR which directly modulates ER-Ca2+ (Ca2+ER), as monitored by a novel ER targeted Ca2+-sensor. Moreover, we validated the Ca2+ex dependent colocalizations and interactions of CaSR with ER-protein processing chaperone, 78-kDa glucose regulated protein (GRP78), and with trafficking-related protein. Live cell imaging results indicated that CaSR and vesicle-associated membrane protein-associated A (VAPA) are inter-dependent during Ca2+ex induced enhancement of near-cell membrane expression. This study significantly extends the repertoire of the CaSR interactome and reveals likely novel players and pathways of CaSR participating in Ca2+ER dynamics, agonist mediated ER-protein processing and surface expression.

Cell line construction and maintenance for Lyso-IP and Endo-IP analysis of amyloid precursor protein processing v1

Lyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach for purification of early/sorting endosomes (Endo-IP). In addition, we have found that endolysosomal purification via Lyso-IP and Endo-IP can be coupled with a quantitative proteomics workflow to obtain snapshots of Amyloid Precursor Protein (APP) processing to its Aβ products (Park et al. in submission). Here, we describe methods for cell line construction and maintenance of 293 cells with TMEM192-3xHA and 3xFLAG-EEA1, which are used for lysosome and endosome purification, respectively, with the addition of patient mutations to APP promotes processing. Cells with endogenously tagged TMEM192 and stably expressing FLAG-EEA1 are referred to as 293EL cells, for Endo-IP and Lyso-IP. These cells were also prepared in a form that has a deletion of the APP gene (293EL;APP-/-) and the same cells reconstituted with a lentivirus stably expressing APPSw;T700N to allow functional analysis of APP processing.