RESUMEN
Cell surface proteins carrying N-glycans play important roles in inter- and intracellular processes including cell adhesion, development, and cellular recognition. Dysregulation of the glycosylation machinery has been implicated in various diseases, and investigation of global differential cell surface proteome effects due to the loss of N-glycosylation will provide comprehensive insights into their pathogenesis. Cell surface proteins isolated from Parent Pro-5 CHO cells (W5 cells), two CHO mutants with loss of N-glycosylation function derived from Pro-5 CHO (Lec1 and Lec4 cells), were subjected to proteome analysis via high-resolution LCMS. We identified 44 and 43 differentially expressed membrane proteins in Lec1 and Lec4 cells, respectively, as compared to W5 cells. The defective N-glycosylation mutants showed increased abundance of integrin subunits in Lec1 and Lec4 cells at the cell surface. We also found significantly reduced levels of IGF-1R (Insulin like growth factor-1 receptor); a receptor tyrosine kinase; and the GTPase activating protein IQGAP1 (IQ motif-containing GTPase activating protein), a highly conserved cytoplasmic scaffold protein) in Lec1 and Lec4 cells. In silico docking studies showed that the IQ domain of IQGAP1 interacts with the kinase domain of IGF-1R. The integrin signaling and insulin growth factor receptor signaling were also enriched according to GSEA analysis and pathway analysis of differentially expressed proteins. Significant reductions of phosphorylation of ERK1 and ERK2 in Lec1 and Lec4 cells were observed upon IGF-1R ligand (IGF-1 LR3) stimulation. IGF-1 LR3, known as Long arginine3-IGF-1, is a synthetic protein and lengthened analog of insulin-like growth factor 1. The work suggests a novel mechanism for the activation of IGF-1 dependent ERK signaling in CHO cells, wherein IQGAP1 plausibly functions as an IGF-1R-associated scaffold protein. Appropriate glycosylation by the enzymes MGAT1 and MGAT5 is thus essential for processing of cell surface receptor IGF-1R, a potential binding partner in IQGAP1 and ERK signaling, the integral components of the IGF pathway.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina , Animales , Cricetinae , Células CHO , Cricetulus , Proteínas Activadoras de GTPasa/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Integrinas/metabolismo , Fosforilación , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , GlicosilaciónRESUMEN
Coronavirus disease (COVID-19) is an acute infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human SP-D (surfactant protein D) is known to interact with the spike protein of SARS-CoV, but its immune surveillance against SARS-CoV-2 is not known. The current study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2. The interaction of rfhSP-D with the spike protein of SARS-CoV-2 and human ACE-2 (angiotensin-converting enzyme 2) receptor was predicted via docking analysis. The inhibition of interaction between the spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was assessed by measuring the expression of RdRp gene of the virus using quantitative PCR. In silico interaction studies indicated that three amino acid residues in the receptor-binding domain of spike protein of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2-positive cases (asymptomatic, n = 7; symptomatic, n = 8) and negative control samples (n = 15) demonstrated that treatment with 1.67 µM rfhSP-D inhibited viral replication by â¼5.5-fold and was more efficient than remdesivir (100 µM) in Vero cells. An approximately two-fold reduction in viral infectivity was also observed after treatment with 1.67 µM rfhSP-D. These results conclusively demonstrate that the rfhSP-D mediated calcium independent interaction between the receptor-binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and significantly reduced SARS-CoV-2 infection and replication in vitro.
Asunto(s)
COVID-19/metabolismo , Proteína D Asociada a Surfactante Pulmonar , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus , Replicación Viral , Adulto , Animales , Chlorocebus aethiops , Femenino , Humanos , Masculino , Unión Proteica , Proteína D Asociada a Surfactante Pulmonar/química , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
The innate immune system is comprised of both cellular and humoral players that recognise and eradicate invading pathogens. Therefore, the interplay between retroviruses and innate immunity has emerged as an important component of viral pathogenesis. HIV-1 infection in humans that results in hematologic abnormalities and immune suppression is well represented by changes in the CD4/CD8 T cell ratio and consequent cell death causing CD4 lymphopenia. The innate immune responses by mucosal barriers such as complement, DCs, macrophages, and NK cells as well as cytokine/chemokine profiles attain great importance in acute HIV-1 infection, and thus, prevent mucosal capture and transmission of HIV-1. Conversely, HIV-1 has evolved to overcome innate immune responses through RNA-mediated rapid mutations, pathogen-associated molecular patterns (PAMPs) modification, down-regulation of NK cell activity and complement receptors, resulting in increased secretion of inflammatory factors. Consequently, epithelial tissues lining up female reproductive tract express innate immune sensors including anti-microbial peptides responsible for forming primary barriers and have displayed an effective potent anti-HIV activity during phase I/II clinical trials.
Asunto(s)
Infecciones por VIH , Seropositividad para VIH , VIH-1 , Femenino , Genitales Femeninos , Humanos , Inmunidad InnataRESUMEN
PROBLEM: Early-onset preeclampsia (EOPE) is a severe gestational hypertensive disorder with significant feto-maternal morbidity and mortality due to uteroplacental insufficiency. Circulating extracellular vesicles of placental origin (EV-P) are known to be involved in the pathophysiology of EOPE and might serve as an ideal reservoir for its specific biomarkers. Therefore, we aimed to characterize and perform comparative proteomics of circulating EV-P from healthy pregnant and EOPE women before delivery. METHOD OF STUDY: The EV-P from both groups were isolated using immunoaffinity and were characterized using transmission electron microscopy, dynamic light scattering, nanoparticle tracking analysis, and immunoblotting. Following IgG albumin depletion, the pooled proteins that were isolated from EV-P of both groups were subjected to quantitative TMT proteomics. RESULTS: Circulating term EV-P isolated from both groups revealed â¼150 nm spherical vesicles containing CD9 and CD63 along with placental PLAP and HLA-G proteins. Additionally, the concentration of EOPE-derived EV-P was significantly increased. A total of 208 proteins were identified, with 26 among them being differentially abundant in EV-P of EOPE women. This study linked the pathophysiology of EOPE to 19 known and seven novel proteins associated with innate immune responses such as complement and TLR signaling along with hemostasis and oxygen homeostasis. CONCLUSION: The theory suggesting circulating EVs of placental origin could mimic molecular information from the parent organ-"the placenta"-is strengthened by this study. The findings pave the way for possible discovery of novel prognostic and predictive biomarkers as well as provide insight into the mechanisms driving the pathogenesis of EOPE.
Asunto(s)
Vesículas Extracelulares , Hemostasis , Inmunidad Innata , Placenta , Preeclampsia , Proteómica , Humanos , Femenino , Embarazo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/inmunología , Preeclampsia/inmunología , Preeclampsia/metabolismo , Adulto , Placenta/metabolismo , Placenta/inmunología , Biomarcadores/metabolismoRESUMEN
PROBLEM: Surfactant protein D (SP-D), a multimeric collectin expressed by testicular mucosal epithelia and is positively regulated by testosterone. It exerts antimicrobial effects, modulates inflammation and rescued spermatogenesis in a murine model. Various cytokines and chemokines, including MCP-1, play a key role in regulating the inflammation in rat and human testis. The study aimed to investigate the role of SP-D and involvement of chemokines and cytokines in the male infertility associated with urogenital infections or inflammation. METHOD OF STUDY: The cross-sectional study evaluated levels of SP-D, testosterone, estradiol and the cytokines/chemokines including MCP-1 in the serum and semen samples of fertile and infertile Indian men with and without urogenital infections/inflammation (n = 76). RESULTS: Both fertile and infertile males with urogenital infection/inflammation had significantly lower levels of SP-D and higher levels of the chemokine, Monocyte chemoattractant protein 1 (MCP-1) in the serum and seminal plasma. Seminal plasma of these males exhibited significantly higher proportion of proteolytically degraded forms of SP-D. The serum SP-D levels positively correlated with testosterone/estradiol (TE) ratio. There was no significant correlation between the SP-D levels in seminal plasma and sperm count/motility. With a significant area under the Receiver Operating Characteristic curves, the serum and seminal plasma SP-D levels exhibited significant potential to predict infertility with high sensitivity and specificity in men with genital infections/inflammation. CONCLUSIONS: The circulating and seminal plasma SP-D levels were decreased in men with urogenital infection and inflammation. This could be due to their engagement at the site of infection, dysregulated expression owing to the altered hormonal profile and increased proteolytic degradation.