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Cell Nov 2023To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization,...
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
Topics: Animals; Male; Mice; Axoneme; Cryoelectron Microscopy; Flagella; Microtubules; Semen; Spermatozoa; Proteome
PubMed: 37865089
DOI: 10.1016/j.cell.2023.09.017 -
Experimental Biology and Medicine... Aug 2023Ubiquitination is an intricately regulated post-translational modification that involves the covalent attachment of ubiquitin to a substrate protein. The complex dynamic... (Review)
Review
Ubiquitination is an intricately regulated post-translational modification that involves the covalent attachment of ubiquitin to a substrate protein. The complex dynamic nature of the ubiquitination process regulates diverse cellular functions including targeting proteins for degradation, cell cycle, deoxyribonucleic acid (DNA) damage repair, and numerous cell signaling pathways. Ubiquitination also serves as a crucial mechanism in protein quality control. Dysregulation in ubiquitination could result in lethal disease conditions such as cancers and neurodegenerative diseases. Therefore, the ubiquitination cascade has become an attractive target for therapeutic interventions. Enormous efforts have been made to detect ubiquitination involving different detection techniques to better grasp the underlying molecular mechanisms of ubiquitination. This review discusses a wide range of techniques stretching from the simplest assays to real-time assays. This includes western blotting/immunoblotting, fluorescence assays, chemiluminescence assays, spectrophotometric assays, and nanopore sensing assays. This review compares these applications, and the inherent advantages and limitations.
Topics: Ubiquitination; Ubiquitin; Proteins; Protein Processing, Post-Translational; Ubiquitin-Protein Ligases
PubMed: 37787047
DOI: 10.1177/15353702231191186 -
Advanced Science (Weinheim,... Sep 2023Sperm-induced Ca rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca oscillations are...
Sperm-induced Ca rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na /Ca exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14 oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.
Topics: Humans; Male; Calcium; Homeostasis; Oocytes; Semen; Sodium-Calcium Exchanger; Ubiquitination; Animals; Mice; Nucleoside-Triphosphatase
PubMed: 37493331
DOI: 10.1002/advs.202301940 -
Proceedings of the National Academy of... Oct 2023Cancer-associated fibroblasts (CAFs) play vital roles in establishing a suitable tumor microenvironment. In this study, RNA sequencing data revealed that CAFs could...
Cancer-associated fibroblasts (CAFs) play vital roles in establishing a suitable tumor microenvironment. In this study, RNA sequencing data revealed that CAFs could promote cell proliferation, angiogenesis, and ECM reconstitution by binding to integrin families and activating PI3K/AKT pathways in esophageal squamous cell carcinoma (ESCC). The secretions of CAFs play an important role in regulating these biological activities. Among these secretions, we found that MFGE8 is specifically secreted by CAFs in ESCC. Additionally, the secreted MFGE8 protein is essential in CAF-regulated vascularization, tumor proliferation, drug resistance, and metastasis. By binding to Integrin αVβ3/αVβ5 receptors, MFGE8 promotes tumor progression by activating both the PI3K/AKT and ERK/AKT pathways. Interestingly, the biological function of MFGE8 secreted by CAFs fully demonstrated the major role of CAFs in ESCC and its mode of mechanism, showing that MFGE8 could be a driver factor of CAFs in remodeling the tumor environment. In vivo treatment targeting CAFs-secreting MFGE8 or its receptor produced significant inhibitory effects on ESCC growth and metastasis, which provides an approach for the treatment of ESCC.
Topics: Humans; Esophageal Squamous Cell Carcinoma; Cancer-Associated Fibroblasts; Esophageal Neoplasms; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Cell Line, Tumor; Cell Proliferation; Fibroblasts; Tumor Microenvironment; Antigens, Surface; Milk Proteins
PubMed: 37816055
DOI: 10.1073/pnas.2307914120 -
MBio Oct 2023Secreted virulence factors play a critical role in bacterial pathogenesis. Virulence effectors not only help bacteria to overcome the host immune system but also aid in...
Secreted virulence factors play a critical role in bacterial pathogenesis. Virulence effectors not only help bacteria to overcome the host immune system but also aid in establishing infection. , which causes tuberculosis in humans, encodes various virulence effectors. Triggers that modulate the secretion of virulence effectors in are yet to be fully understood. To gain mechanistic insight into the secretion of virulence effectors, we performed high-throughput proteomic studies. With the help of system-level protein-protein interaction network analysis and empirical validations, we unravelled a link between phosphorylation and secretion. Taking the example of the well-known virulence factor of CFP10, we show that the dynamics of CFP10 phosphorylation strongly influenced bacterial virulence and survival and . This study presents the role of phosphorylation in modulating the secretion of virulence factors.
Topics: Humans; Mycobacterium tuberculosis; Bacterial Proteins; Antigens, Bacterial; Phosphorylation; Virulence; Proteomics; Virulence Factors
PubMed: 37791794
DOI: 10.1128/mbio.01232-23 -
Nature Chemical Biology Sep 2023Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been...
Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.
Topics: Animals; Mice; Protein Sorting Signals; Protein Transport; Membrane Proteins; SEC Translocation Channels; Protein Biosynthesis
PubMed: 37169961
DOI: 10.1038/s41589-023-01326-1 -
Adipocyte Dec 2023Adipokines are proteins secreted by adipose tissue to regulate glucolipid metabolism and play vital roles in our body. Different adipokines have more than one endocrine...
Adipokines are proteins secreted by adipose tissue to regulate glucolipid metabolism and play vital roles in our body. Different adipokines have more than one endocrine function and be divided into several different categories according to their functions, including adipokines involved in glucolipid metabolism, the inflammatory response, insulin action, activation of brown adipose tissue (BAT) and appetite regulation. Multiple adipokines interact with each other to regulate metabolic processes. Based on the recent progress of adipokine research, this article discusses the role and mechanism of various adipokines in glucolipid metabolism, which may provide new ideas for understanding the pathogenesis and improving the treatment of various metabolic diseases.
Topics: Adipokines; Glucose; Lipid Metabolism; Adipose Tissue; Adipose Tissue, Brown; Energy Metabolism; Leptin
PubMed: 37077042
DOI: 10.1080/21623945.2023.2202976 -
Cells Jul 2023The proteasome is a multi-catalytic protease complex that is involved in protein quality control via three proteolytic activities (i.e., caspase-, trypsin-, and... (Review)
Review
The proteasome is a multi-catalytic protease complex that is involved in protein quality control via three proteolytic activities (i.e., caspase-, trypsin-, and chymotrypsin-like activities). Most cellular proteins are selectively degraded by the proteasome via ubiquitination. Moreover, the ubiquitin-proteasome system is a critical process for maintaining protein homeostasis. Here, we briefly summarize the structure of the proteasome, its regulatory mechanisms, proteins that regulate proteasome activity, and alterations to proteasome activity found in diverse diseases, chemoresistant cells, and cancer stem cells. Finally, we describe potential therapeutic modalities that use the ubiquitin-proteasome system.
Topics: Proteasome Endopeptidase Complex; Proteolysis; Ubiquitination; Ubiquitin; Proteins
PubMed: 37508510
DOI: 10.3390/cells12141846 -
Journal of Proteome Research Nov 2023Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules...
Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the or gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..
Topics: Humans; Carrier Proteins; Endoplasmic Reticulum; Golgi Apparatus; Membrane Proteins; Protein Transport
PubMed: 37844105
DOI: 10.1021/acs.jproteome.3c00259 -
PLoS Pathogens Oct 2023In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation...
In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.
Topics: Glycosylation; Flavivirus; Viral Envelope Proteins; Encephalitis Virus, Japanese; Virion
PubMed: 37819933
DOI: 10.1371/journal.ppat.1011681