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Cell Metabolism Sep 2022The drivers of sporadic Alzheimer's disease (AD) remain incompletely understood. Utilizing directly converted induced neurons (iNs) from AD-patient-derived fibroblasts,...
The drivers of sporadic Alzheimer's disease (AD) remain incompletely understood. Utilizing directly converted induced neurons (iNs) from AD-patient-derived fibroblasts, we identified a metabolic switch to aerobic glycolysis in AD iNs. Pathological isoform switching of the glycolytic enzyme pyruvate kinase M (PKM) toward the cancer-associated PKM2 isoform conferred metabolic and transcriptional changes in AD iNs. These alterations occurred via PKM2's lack of metabolic activity and via nuclear translocation and association with STAT3 and HIF1α to promote neuronal fate loss and vulnerability. Chemical modulation of PKM2 prevented nuclear translocation, restored a mature neuronal metabolism, reversed AD-specific gene expression changes, and re-activated neuronal resilience against cell death.
Topics: Alzheimer Disease; Glycolysis; Humans; Neoplasms; Protein Isoforms; Pyruvate Kinase
PubMed: 35987203
DOI: 10.1016/j.cmet.2022.07.014 -
Nucleic Acids Research May 2023In situ capturing technologies add tissue context to gene expression data, with the potential of providing a greater understanding of complex biological systems....
In situ capturing technologies add tissue context to gene expression data, with the potential of providing a greater understanding of complex biological systems. However, splicing variants and full-length sequence heterogeneity cannot be characterized at spatial resolution with current transcriptome profiling methods. To that end, we introduce spatial isoform transcriptomics (SiT), an explorative method for characterizing spatial isoform variation and sequence heterogeneity using long-read sequencing. We show in mouse brain how SiT can be used to profile isoform expression and sequence heterogeneity in different areas of the tissue. SiT reveals regional isoform switching of Plp1 gene between different layers of the olfactory bulb, and the use of external single-cell data allows the nomination of cell types expressing each isoform. Furthermore, SiT identifies differential isoform usage for several major genes implicated in brain function (Snap25, Bin1, Gnas) that are independently validated by in situ sequencing. SiT also provides for the first time an in-depth A-to-I RNA editing map of the adult mouse brain. Data exploration can be performed through an online resource (https://www.isomics.eu), where isoform expression and RNA editing can be visualized in a spatial context.
Topics: Animals; Mice; Alternative Splicing; Sequence Analysis, RNA; Protein Isoforms; Gene Expression Profiling; Gene Expression; Transcriptome
PubMed: 36928528
DOI: 10.1093/nar/gkad169 -
Science Advances Jan 2022Tumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is unknown. We developed a long-read (LR)...
Tumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is unknown. We developed a long-read (LR) RNA sequencing and analytical platform that identifies and annotates full-length isoforms and infers tumor-specific splicing events. Application of this platform to breast cancer samples identifies thousands of previously unannotated isoforms; ~30% affect protein coding exons and are predicted to alter protein localization and function. We performed extensive cross-validation with -omics datasets to support transcription and translation of novel isoforms. We identified 3059 breast tumor–specific splicing events, including 35 that are significantly associated with patient survival. Of these, 21 are absent from GENCODE and 10 are enriched in specific breast cancer subtypes. Together, our results demonstrate the complexity, cancer subtype specificity, and clinical relevance of previously unidentified isoforms and splicing events in breast cancer that are only annotatable by LR-seq and provide a rich resource of immuno-oncology therapeutic targets.
Topics: Alternative Splicing; Breast Neoplasms; Female; High-Throughput Nucleotide Sequencing; Humans; Protein Isoforms; Sequence Analysis, RNA; Transcriptome
PubMed: 35044822
DOI: 10.1126/sciadv.abg6711 -
Nature Apr 2023Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein, to trigger target cell pyroptosis. GSDMB and the charter gasdermin...
Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein, to trigger target cell pyroptosis. GSDMB and the charter gasdermin family member GSDMD have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. ). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently. Here we report the crystal structure of the IpaH7.8-GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins. GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies. Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Cryoelectron Microscopy; Crystallography, X-Ray; Gasdermins; Neoplasm Proteins; Pore Forming Cytotoxic Proteins; Protein Isoforms; Pyroptosis; Shigella flexneri; Species Specificity; Alternative Splicing
PubMed: 36991125
DOI: 10.1038/s41586-023-05872-5 -
Nature May 2023Mitotic defects activate the spindle-assembly checkpoint, which inhibits the anaphase-promoting complex co-activator CDC20 to induce a prolonged cell cycle arrest. Once...
Mitotic defects activate the spindle-assembly checkpoint, which inhibits the anaphase-promoting complex co-activator CDC20 to induce a prolonged cell cycle arrest. Once errors are corrected, the spindle-assembly checkpoint is silenced, allowing anaphase onset to occur. However, in the presence of persistent unresolvable errors, cells can undergo 'mitotic slippage', exiting mitosis into a tetraploid G1 state and escaping the cell death that results from a prolonged arrest. The molecular logic that enables cells to balance these duelling mitotic arrest and slippage behaviours remains unclear. Here we demonstrate that human cells modulate the duration of their mitotic arrest through the presence of conserved, alternative CDC20 translational isoforms. Downstream translation initiation results in a truncated CDC20 isoform that is resistant to spindle-assembly-checkpoint-mediated inhibition and promotes mitotic exit even in the presence of mitotic perturbations. Our study supports a model in which the relative levels of CDC20 translational isoforms control the duration of mitotic arrest. During a prolonged mitotic arrest, new protein synthesis and differential CDC20 isoform turnover create a timer, with mitotic exit occurring once the truncated Met43 isoform achieves sufficient levels. Targeted molecular changes or naturally occurring cancer mutations that alter CDC20 isoform ratios or its translational control modulate mitotic arrest duration and anti-mitotic drug sensitivity, with potential implications for the diagnosis and treatment of human cancers.
Topics: Humans; Cdc20 Proteins; Protein Biosynthesis; Protein Isoforms; Spindle Apparatus; Peptide Chain Initiation, Translational; M Phase Cell Cycle Checkpoints
PubMed: 37100900
DOI: 10.1038/s41586-023-05943-7 -
Nature Communications May 2023Isoforms of a gene may contribute to diverse biological functions. In the cochlea, the repertoire of alternative isoforms remains unexplored. We integrated single-cell...
Isoforms of a gene may contribute to diverse biological functions. In the cochlea, the repertoire of alternative isoforms remains unexplored. We integrated single-cell short-read and long-read RNA sequencing techniques and identified 236,012 transcripts, 126,612 of which were unannotated in the GENCODE database. Then we analyzed and verified the unannotated transcripts using RNA-seq, RT-PCR, Sanger sequencing, and MS-based proteomics approaches. To illustrate the importance of identifying spliced isoforms, we investigated otoferlin, a key protein involved in synaptic transmission in inner hair cells (IHCs). Upon deletion of the canonical otoferlin isoform, the identified short isoform is able to support normal hearing thresholds but with reduced sustained exocytosis of IHCs, and further revealed otoferlin functions in endocytic membrane retrieval that was not well-addressed previously. Furthermore, we found that otoferlin isoforms are associated with IHC functions and auditory phenotypes. This work expands our mechanistic understanding of auditory functions at the level of isoform resolution.
Topics: Mice; Animals; Mice, Knockout; Exocytosis; Hair Cells, Auditory, Inner; Hearing; Protein Isoforms; Cochlea; Synapses; Membrane Proteins
PubMed: 37248244
DOI: 10.1038/s41467-023-38621-3 -
Nucleic Acids Research Jan 2022APPRIS (https://appris.bioinfo.cnio.es) is a well-established database housing annotations for protein isoforms for a range of species. APPRIS selects principal...
APPRIS (https://appris.bioinfo.cnio.es) is a well-established database housing annotations for protein isoforms for a range of species. APPRIS selects principal isoforms based on protein structure and function features and on cross-species conservation. Most coding genes produce a single main protein isoform and the principal isoforms chosen by the APPRIS database best represent this main cellular isoform. Human genetic data, experimental protein evidence and the distribution of clinical variants all support the relevance of APPRIS principal isoforms. APPRIS annotations and principal isoforms have now been expanded to 10 model organisms. In this paper we highlight the most recent updates to the database. APPRIS annotations have been generated for two new species, cow and chicken, the protein structural information has been augmented with reliable models from the EMBL-EBI AlphaFold database, and we have substantially expanded the confirmatory proteomics evidence available for the human genome. The most significant change in APPRIS has been the implementation of TRIFID functional isoform scores. TRIFID functional scores are assigned to all splice isoforms, and APPRIS uses the TRIFID functional scores and proteomics evidence to determine principal isoforms when core methods cannot.
Topics: Animals; Cattle; Chickens; Databases, Protein; Humans; Protein Conformation; Protein Isoforms; Proteins; Proteomics
PubMed: 34755885
DOI: 10.1093/nar/gkab1058 -
Annual Review of Biomedical Data Science Aug 2023Alternative splicing is pivotal to the regulation of gene expression and protein diversity in eukaryotic cells. The detection of alternative splicing events requires... (Review)
Review
Alternative splicing is pivotal to the regulation of gene expression and protein diversity in eukaryotic cells. The detection of alternative splicing events requires specific omics technologies. Although short-read RNA sequencing has successfully supported a plethora of investigations on alternative splicing, the emerging technologies of long-read RNA sequencing and top-down mass spectrometry open new opportunities to identify alternative splicing and protein isoforms with less ambiguity. Here, we summarize improvements in short-read RNA sequencing for alternative splicing analysis, including percent splicing index estimation and differential analysis. We also review the computational methods used in top-down proteomics analysis regarding proteoform identification, including the construction of databases of protein isoforms and statistical analyses of search results. While many improvements in sequencing and computational methods will result from emerging technologies, there should be future endeavors to increase the effectiveness, integration, and proteome coverage of alternative splicing events.
Topics: Proteomics; Transcriptome; Protein Isoforms; Alternative Splicing; RNA Splicing
PubMed: 37561601
DOI: 10.1146/annurev-biodatasci-020722-044021 -
Advances in Clinical Chemistry 2018Adiponectin circulates in blood in multiple isoforms. High molecular weight (HMW) adiponectin is thought to be most biologically active and promotes glucose uptake,... (Review)
Review
Adiponectin circulates in blood in multiple isoforms. High molecular weight (HMW) adiponectin is thought to be most biologically active and promotes glucose uptake, insulin sensitivity, and fatty acid oxidation. In obesity, adiponectin isoform formation is disrupted, leading to an inverse association between metabolic disease and HMW and total adiponectin. Adiponectin isoforms also function as acute-phase reactants influencing inflammation in acute and chronic disease. Interestingly, adiponectin and mortality have a U-shaped association. Unfortunately, data concerning adiponectin and its pathophysiologic function conflict. This is predominantly due to difficulties in adequate measurement of adiponectin isoforms and lack of a gold standard. In this review we provide a general overview of the formation and function of adiponectin and its isoforms under physiologic conditions. We highlight the ways adiponectin isoform formation is disrupted in obesity and its ensuing pathologic conditions. Furthermore, we will elaborate on the role of adiponectin isoforms as inflammatory proteins with respect to cardiac and kidney disease and discuss the association of adiponectin with mortality. Finally, we will provide a historical perspective on the measurement of adiponectin isoforms, current limitations, and future challenges.
Topics: Adiponectin; Animals; Cardiovascular Diseases; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Obesity; Protein Isoforms
PubMed: 29655459
DOI: 10.1016/bs.acc.2018.02.007 -
Biomolecules Aug 2022The heat shock protein 90 (Hsp90) is a molecular chaperone and a key regulator of proteostasis under both physiological and stress conditions. In mammals, there are two... (Review)
Review
The heat shock protein 90 (Hsp90) is a molecular chaperone and a key regulator of proteostasis under both physiological and stress conditions. In mammals, there are two cytosolic Hsp90 isoforms: Hsp90α and Hsp90β. These two isoforms are 85% identical and encoded by two different genes. Hsp90β is constitutively expressed and essential for early mouse development, while Hsp90α is stress-inducible and not necessary for survivability. These two isoforms are known to have largely overlapping functions and to interact with a large fraction of the proteome. To what extent there are isoform-specific functions at the protein level has only relatively recently begun to emerge. There are studies indicating that one isoform is more involved in the functionality of a specific tissue or cell type. Moreover, in many diseases, functionally altered cells appear to be more dependent on one particular isoform. This leaves space for designing therapeutic strategies in an isoform-specific way, which may overcome the unfavorable outcome of pan-Hsp90 inhibition encountered in previous clinical trials. For this to succeed, isoform-specific functions must be understood in more detail. In this review, we summarize the available information on isoform-specific functions of mammalian Hsp90 and connect it to possible clinical applications.
Topics: Animals; HSP90 Heat-Shock Proteins; Mice; Molecular Chaperones; Protein Isoforms; Proteome
PubMed: 36139005
DOI: 10.3390/biom12091166