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Cells Jun 2024Inflammasomes comprise a group of protein complexes with fundamental roles in the induction of inflammation. Upon sensing stress factors, their assembly induces the... (Review)
Review
Inflammasomes comprise a group of protein complexes with fundamental roles in the induction of inflammation. Upon sensing stress factors, their assembly induces the activation and release of the pro-inflammatory cytokines interleukin (IL)-1β and -18 and a lytic type of cell death, termed pyroptosis. Recently, CARD8 has joined the group of inflammasome sensors. The carboxy-terminal part of CARD8, consisting of a function-to-find-domain (FIIND) and a caspase activation and recruitment domain (CARD), resembles that of NLR family pyrin domain containing 1 (NLRP1), which is recognized as the main inflammasome sensor in human keratinocytes. The interaction with dipeptidyl peptidases 8 and 9 (DPP8/9) represents an activation checkpoint for both sensors. CARD8 and NLRP1 are activated by viral protease activity targeting their amino-terminal region. However, CARD8 also has some unique features compared to the established inflammasome sensors. Activation of CARD8 occurs independently of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), leading mainly to pyroptosis rather than the activation and secretion of pro-inflammatory cytokines. CARD8 was also shown to have anti-inflammatory and anti-apoptotic activity. It interacts with, and inhibits, several proteins involved in inflammation and cell death, such as the inflammasome sensor NLRP3, CARD-containing proteins caspase-1 and -9, nucleotide-binding oligomerization domain containing 2 (NOD2), or nuclear factor kappa B (NF-κB). Single nucleotide polymorphisms (SNPs) of , some of them occurring at high frequencies, are associated with various inflammatory diseases. The molecular mechanisms underlying the different pro- and anti-inflammatory activities of CARD8 are incompletely understood. Alternative splicing leads to the generation of multiple CARD8 protein isoforms. Although the functional properties of these isoforms are poorly characterized, there is evidence that suggests isoform-specific roles. The characterization of the functions of these isoforms, together with their cell- and disease-specific expression, might be the key to a better understanding of CARD8's different roles in inflammation and inflammatory diseases.
Topics: Humans; Inflammasomes; CARD Signaling Adaptor Proteins; Apoptosis; Inflammation; Animals; Pyroptosis; Anti-Inflammatory Agents; Neoplasm Proteins
PubMed: 38920661
DOI: 10.3390/cells13121032 -
Cells Jun 2024Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is...
Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the splice variants (, ) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the gene on the expression of and was also investigated. The TID proteins and the expression of splice variants were detected. After differentiation, the expression of and increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of . Three days after transfection, the expression of increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of and changes under differentiation of B-MSCs into osteoblasts and may influence the expression of . However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.
Topics: Humans; Osteoblasts; Mesenchymal Stem Cells; Cell Differentiation; Osteogenesis; Protein Isoforms; Alkaline Phosphatase; Bone Marrow Cells; Cell Proliferation
PubMed: 38920651
DOI: 10.3390/cells13121021 -
Acta Chimica Slovenica Apr 2024Breast cancer cell growth is often dependent on the presence of steroidal hormones. The 17β-hydroxysteroid dehydrogenase type 1 isoform (17βHSD1) catalyzes...
Breast cancer cell growth is often dependent on the presence of steroidal hormones. The 17β-hydroxysteroid dehydrogenase type 1 isoform (17βHSD1) catalyzes NADPH-dependent conversion of estrone to estradiol, a more potent estrogen, and represents potential drug target for breast cancer treatment. To provide active enzyme for inhibitor screening, 17βHSD1 is usually expressed in insect or mammalian cells, or isolated from human placenta. In the present study we describe a simple protocol for expression and purification of active human 17βHSD1 from BL21(DE3) Escherichia coli cells. Soluble human 17βHSD1 was expressed using a pET28a(+)-based plasmid, which encodes a hexahistidine tag fused to the N-terminus of the protein, and purified by nickel affinity chromatography. The enzyme activity of purified 17βHSD1 was verified by three methods: thin-layer chromatography, an alkali assay and a spectroscopic assay. These non-radioactive enzyme assays require only standard laboratory equipment, and can be used for screening compounds that modulate 17βHSD1 activity.
Topics: Humans; Escherichia coli; 17-Hydroxysteroid Dehydrogenases; Chromatography, Affinity; Recombinant Proteins
PubMed: 38919102
DOI: 10.17344/acsi.2024.8629 -
Asian Pacific Journal of Cancer... Jun 2024Cytochrome P450 (CYP) are phase I metabolizing enzymes involved in detoxification of chemotherapeutic agents. Among the CYP gene family, including CYP1A1, CYP1B1, CYP2C,...
BACKGROUND
Cytochrome P450 (CYP) are phase I metabolizing enzymes involved in detoxification of chemotherapeutic agents. Among the CYP gene family, including CYP1A1, CYP1B1, CYP2C, CYP2D, CYP2E and CYP17, their significance in cancer susceptibility is well established. However, there remains limited understanding regarding the polymorphisms of CYP2C19*2 and CYP17 and their potential correlation with chemotherapy-induced toxicity reactions in breast cancer (BC) patients. In this study we intended to identify the association of CYP2C19*2 and CYP17 gene polymorphisms on drug response as well as toxicity reactions in BC patients undergoing adriamycin/paclitaxel based chemotherapy within Indian population.
METHODS
Two hundred BC patients receiving adriamycin and paclitaxel chemotherapy were enrolled in this study and chemotherapy induced hematological and non-hematological toxicity reactions were noted. The polymorphisms of CYP2C19*2 (681G>A) and CYP17 (34T>C) isoforms of cytochrome p 450 gene was studied by PCR and RFLP analysis.
RESULTS
The univariate logistic regression analysis revealed significant associations between CYP2C19*2 (681 G>A) polymorphisms with hematological toxicities i.e., anemia (OR=9.77, 95% CI: 2.84-33.52; p=0.0003), neutropenia (OR=5.72, 95% CI: 1.75-18.68; p=0.003), febrile neutropenia (OR=4.29, 95% CI: 1.32-13.87; p=0.014) and thrombocytopenia (OR=5.86, 95% CI: 1.15-29.72); p=0.032) in BC patients. Additionally BC patients treated with adriamycin exhibited significant association between CYP2C19*2 polymorphism with chemotherapy induced nausea and vomiting (CINV) (OR=99.73, 95% CI: 5.70-174.64); p=0.001), fatigue (OR=83.29, 95% CI: 4.77-145.69); p=0.002), bodyache (OR=4.44, 95% CI: 1.24-15.91); p=0.021) and peripheral neuropathy (OR=12.00, 95% CI: 1.80-79.89); p=0.010. Furthermore, the regression analysis indicated an association between CYP17 with body ache (OR=2.77, 95% CI: 1.21-6.34; p=0.015) and peripheral neuropathy (OR=3.90, 95% CI: 1.59-9.53; p=0.002) in BC patients treated with paclitaxel chemotherapy.
CONCLUSION
The findings obtained from this study illustrated significant association of CYP2C9*2 (681G>A) polymorphism with adreamicin based chemotherapy induced toxicities and CYP17 (34T>C) polymorphism with paclitaxel induced bodyache and peripheral neuropathy in BC patients.
Topics: Humans; Female; Breast Neoplasms; Paclitaxel; Doxorubicin; Cytochrome P-450 CYP2C19; Middle Aged; Antineoplastic Combined Chemotherapy Protocols; Polymorphism, Single Nucleotide; Adult; Steroid 17-alpha-Hydroxylase; Prognosis; Follow-Up Studies; Aged
PubMed: 38918659
DOI: 10.31557/APJCP.2024.25.6.1977 -
Molecular Systems Biology Jun 2024The variability of proteins at the sequence level creates an enormous potential for proteome complexity. Exploring the depths and limits of this complexity is an ongoing...
The variability of proteins at the sequence level creates an enormous potential for proteome complexity. Exploring the depths and limits of this complexity is an ongoing goal in biology. Here, we systematically survey human and plant high-throughput bottom-up native proteomics data for protein truncation variants, where substantial regions of the full-length protein are missing from an observed protein product. In humans, Arabidopsis, and the green alga Chlamydomonas, approximately one percent of observed proteins show a short form, which we can assign by comparison to RNA isoforms as either likely deriving from transcript-directed processes or limited proteolysis. While some detected protein fragments align with known splice forms and protein cleavage events, multiple examples are previously undescribed, such as our observation of fibrocystin proteolysis and nuclear translocation in a green alga. We find that truncations occur almost entirely between structured protein domains, even when short forms are derived from transcript variants. Intriguingly, multiple endogenous protein truncations of phase-separating translational proteins resemble cleaved proteoforms produced by enteroviruses during infection. Some truncated proteins are also observed in both humans and plants, suggesting that they date to the last eukaryotic common ancestor. Finally, we describe novel proteoform-specific protein complexes, where the loss of a domain may accompany complex formation.
PubMed: 38918600
DOI: 10.1038/s44320-024-00048-3 -
Communications Biology Jun 2024The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub...
The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the β hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the β holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αβ heterooligomers for their dynamic localization within synapses in neurons.
Topics: Cryoelectron Microscopy; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Molecular Dynamics Simulation; Humans; Holoenzymes; Protein Multimerization; Animals
PubMed: 38918547
DOI: 10.1038/s42003-024-06423-y -
Plant Physiology and Biochemistry : PPB Jun 2024Alternative splicing enhances diversity at the transcriptional and protein levels that widely involved in plant response to biotic and abiotic stresses. V. amurensis is...
Alternative splicing enhances diversity at the transcriptional and protein levels that widely involved in plant response to biotic and abiotic stresses. V. amurensis is an extremely cold-tolerant wild grape variety, however, studies on alternative splicing (AS) in amur grape at low temperatures are currently poorly understood. In this study, we analyzed full-length transcriptome and RNA seq data at 0, 2, and 24 h after cold stress in V. amurensis roots. Following quality control and correction, 221,170 high-quality full-length non-concatemer (FLNC) reads were identified. A total of 16,181 loci and 30,733 isoforms were identified. These included 22,868 novel isoforms from annotated genes and 2815 isoforms from 2389 novel genes. Among the distinguished novel isoforms, 673 Long non-coding RNAs (LncRNAs) and 18,164 novel isoforms open reading frame (ORF) region were found. A total of 2958 genes produced 8797 AS events, of which 189 genes were involved in the low-temperature response. Twelve transcription factors show AS during cold treatment and VaMYB108 was selected for initial exploration. Two transcripts, Chr05.63.1 (VaMYB108) and Chr05.63.2 (VaMYB108) of VaMYB108, display up-regulated expression after cold treatment in amur grape roots and are both localized in the nucleus. Only VaMYB108 exhibits transcriptional activation activity. Overexpression of either VaMYB108 or VaMYB108 in grape roots leads to increased expression of the other transcript and both increased chilling resistance of amur grape roots. The results improve and supplement the genome annotations and provide insights for further investigation into AS mechanisms during cold stress in V. amurensis.
PubMed: 38917739
DOI: 10.1016/j.plaphy.2024.108863 -
Nucleic Acids Research Jun 20243'Untranslated regions (3'UTRs) are essential portions of genes containing elements necessary for pre-mRNA 3'end processing and are involved in post-transcriptional gene...
3'Untranslated regions (3'UTRs) are essential portions of genes containing elements necessary for pre-mRNA 3'end processing and are involved in post-transcriptional gene regulation. Despite their importance, they remain poorly characterized in eukaryotes. Here, we have used a multi-pronged approach to extract and curate 3'UTR data from 11533 publicly available datasets, corresponding to the entire collection of Caenorhabditis elegans transcriptomes stored in the NCBI repository from 2009 to 2023. We have also performed high throughput cloning pipelines to identify and validate rare 3'UTR isoforms and incorporated and manually curated 3'UTR isoforms from previously published datasets. This updated C. elegans 3'UTRome (v3) is the most comprehensive resource in any metazoan to date, covering 97.4% of the 20362 experimentally validated protein-coding genes with refined and updated 3'UTR boundaries for 23489 3'UTR isoforms. We also used this novel dataset to identify and characterize sequence elements involved in pre-mRNA 3'end processing and update miRNA target predictions. This resource provides important insights into the 3'UTR formation, function, and regulation in eukaryotes.
PubMed: 38917330
DOI: 10.1093/nar/gkae543 -
Proceedings of the National Academy of... Jul 2024S100A1, a small homodimeric EF-hand Ca-binding protein (~21 kDa), plays an important regulatory role in Ca signaling pathways involved in various biological functions...
S100A1, a small homodimeric EF-hand Ca-binding protein (~21 kDa), plays an important regulatory role in Ca signaling pathways involved in various biological functions including Ca cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca. Ca-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders.
Topics: Ryanodine Receptor Calcium Release Channel; S100 Proteins; Calcium; Cryoelectron Microscopy; Animals; Protein Binding; Binding Sites; Models, Molecular; Protein Conformation; Humans
PubMed: 38917010
DOI: 10.1073/pnas.2400497121 -
BioRxiv : the Preprint Server For... Jun 2024Studying protein isoforms is an essential step in biomedical research; at present, the main approach for analyzing proteins is via bottom-up mass spectrometry...
Studying protein isoforms is an essential step in biomedical research; at present, the main approach for analyzing proteins is via bottom-up mass spectrometry proteomics, which return peptide identifications, that are indirectly used to infer the presence of protein isoforms. However, the detection and quantification processes are noisy; in particular, peptides may be erroneously detected, and most peptides, known as shared peptides, are associated to multiple protein isoforms. As a consequence, studying individual protein isoforms is challenging, and inferred protein results are often abstracted to the gene-level or to groups of protein isoforms. Here, we introduce , a novel statistical method to perform inference at the isoform level. Our method enhances the information available, by integrating mass spectrometry proteomics and transcriptomics data in a Bayesian probabilistic framework. To account for the uncertainty in the measurement process, we propose a two-layer latent variable approach: first, we sample if a peptide has been correctly detected (or, alternatively filter peptides); second, we allocate the abundance of such selected peptides across the protein(s) they are compatible with. This enables us, starting from peptide-level data, to recover protein-level data; in particular, we: i) infer the presence/absence of each protein isoform (via a posterior probability), ii) estimate its abundance (and credible interval), and iii) target isoforms where transcript and protein relative abundances significantly differ. We benchmarked our approach in simulations, and in two multi-protease real datasets: our method displays good sensitivity and specificity when detecting protein isoforms, its estimated abundances highly correlate with the ground truth, and can detect changes between protein and transcript relative abundances. is freely distributed as a Bioconductor R package, and is accompanied by an example usage vignette.
PubMed: 38915658
DOI: 10.1101/2024.06.10.598223