Mitochondria form a dynamic network that responds to physiological signals and metabolic stresses by altering the balance between fusion and fission. Mitochondrial fusion is orchestrated by conserved ...GTPases MFN1/2 and OPA1, a process coordinated in yeast by Ugo1, a mitochondrial metabolite carrier family protein. We uncovered a homozygous missense mutation in SLC25A46, the mammalian orthologue of Ugo1, in a subject with Leigh syndrome. SLC25A46 is an integral outer membrane protein that interacts with MFN2, OPA1, and the mitochondrial contact site and cristae organizing system (MICOS) complex. The subject mutation destabilizes the protein, leading to mitochondrial hyperfusion, alterations in endoplasmic reticulum (ER) morphology, impaired cellular respiration, and premature cellular senescence. The MICOS complex is disrupted in subject fibroblasts, resulting in strikingly abnormal mitochondrial architecture, with markedly shortened cristae. SLC25A46 also interacts with the ER membrane protein complex EMC, and phospholipid composition is altered in subject mitochondria. These results show that SLC25A46 plays a role in a mitochondrial/ER pathway that facilitates lipid transfer, and link altered mitochondrial dynamics to early‐onset neurodegenerative disease and cell fate decisions.
Synopsis
Whole‐exome sequencing in a Leigh syndrome patient identified mutations in SLC25A46, a degenerate member of the mitochondrial metabolite transport family, linking altered mitochondrial dynamics to early‐onset neurodegenerative disease.
Loss of SLC25A46 results in mitochondrial hyperfusion and striking changes in mitochondrial architecture.
SLC25A46 is an outer membrane protein that interacts with MFN2, OPA1, the MICOS complex, and the EMC complex in the ER.
Loss of SLC25A46 results in altered ER morphology and marked changes in the phospholipid composition of the mitochondrial membranes.
Loss of SLC25A46 results in premature cellular senescence in dividing cells.
Whole‐exome sequencing in a Leigh syndrome patient identified mutations in SLC25A46, a degenerate member of the mitochondrial metabolite transport family, linking altered mitochondrial dynamics to early‐onset neurodegenerative disease.
Alternative splicing (AS) generates extensive transcriptomic and proteomic complexity. However, the functions of species- and lineage-specific splice variants are largely unknown. Here we show that ...mammalian-specific skipping of polypyrimidine tract–binding protein 1 (PTBP1) exon 9 alters the splicing regulatory activities of PTBP1 and affects the inclusion levels of numerous exons. During neurogenesis, skipping of exon 9 reduces PTBP1 repressive activity so as to facilitate activation of a brain-specific AS program. Engineered skipping of the orthologous exon in chicken cells induces a large number of mammalian-like AS changes in PTBP1 target exons. These results thus reveal that a single exon-skipping event in an RNA binding regulator directs numerous AS changes between species. Our results further suggest that these changes contributed to evolutionary differences in the formation of vertebrate nervous systems.
Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. ...Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.
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•Large-scale pooled tethering assay identifies ∼250 transcriptional activators in humans•Fragment screen pinpoints novel activation domains among screen hits•Proteomics, chemical inhibitors, and AlphaFold connect activators to their cofactors•Functional characterization of SRF-C3orf62, an oncogenic fusion found in myofibroma
Alerasool et al. use a pooled assay to identify over 200 transcriptional activators in the human proteome. They also employ fragment screens to identify short activation domains among the hits, connect activators by proximity biotinylation and AlphaFold predictions to distinct co-activators, and functionally characterize several previously unknown transcriptional activators.
We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction ...network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles.
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•We created a high-resolution human mitochondrial protein proximity map using BioID•Bait-bait analysis shows that the map has sub-compartment resolution•Prey correlation analysis identifies functional clusters and specific modules•OMM baits show specific interactions reflecting contact sites and dual localization
Antonicka et al. used BioID, a proximity-dependent biotinylation assay, to create a high-density mitochondrial proximity interaction map. They were able to identify about half of the mitochondrial proteome, assign proteins to functional clusters and specific modules, show dual protein localization, and demonstrate specific interactions with other cellular organelles.
The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of ...1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses.
Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors ...(cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement.
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•Client interactions for >60 chaperones and cochaperones mapped by AP-MS and LUMIER•Characterization of cellular roles of cochaperones and their client specificities•NUDC family cochaperones associate with β-propeller domains (Kelch, WD40, and RCC1)•Cochaperones may promote the evolutionary diversification of client folds
Mass spectrometry and quantitative LUMIER assays map the proteostasis network in human cells, revealing hundreds of new client proteins, their integration into the network, and the client specificity of most cochaperones.
Pseudouridylation is a common post‐transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity‐biotinylation assay, we ...identified a protein module in mitochondrial RNA granules, platforms for post‐transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine‐Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival.
Synopsis
Using a proximity biotinylation assay, the authors identify a pseudouridine synthase module in mitochondrial RNA granules that is essential for epigenetic modification of the mitochondrial transcriptome, ribosome biogenesis, and mitochondrial protein synthesis.
Mitochondrial RNA granules contain a pseudouridine synthase module.
Pseudouridylation of 16S rRNA by RPUSD4 is essential for mitochondrial ribosome assembly.
Pseudouridylation of specific mitochondrial mRNAs by TRUB2/RPUSD3 is necessary for their translation.
Epitranscriptomic modification of mitochondrial RNA is thus essential for cell viability.
Using a proximity biotinylation assay, the authors identify a pseudouridine synthase module in mitochondrial RNA granules that is essential for epigenetic modification of the mitochondrial transcriptome, ribosome biogenesis, and mitochondrial protein synthesis.
Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited ...proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.
There is increasing evidence that hexokinase is involved in cell proliferation and migration. However, the function of the hexokinase domain containing protein‐1 (HKDC1) in gastric cancer (GC) ...remains unclear. Immunohistochemistry analysis and big data mining were used to evaluate the correlation between HKDC1 expression and clinical features in GC. In addition, the biological function and molecular mechanism of HKDC1 in GC were studied by in vitro and in vivo assays. Our study indicated that HKDC1 expression was upregulated in GC tissues compared with adjacent nontumor tissues. High expression of HKDC1 was associated with worse prognosis. Functional experiments demonstrated that HKDC1 upregulation promoted glycolysis, cell proliferation, and tumorigenesis. In addition, HKDC1 could enhance GC invasion and metastasis by inducing epithelial–mesenchymal transition (EMT). Abrogation of HKDC1 could effectively attenuate its oncogenic and metastatic function. Moreover, HKDC1 promoted GC proliferation and migration in vivo. HKDC1 overexpression conferred chemoresistance to cisplatin, oxaliplatin, and 5‐fluorouracil (5‐Fu) onto GC cells. Furthermore, nuclear factor kappa‐B (NF‐κB) inhibitor PS‐341 could attenuate tumorigenesis, metastasis, and drug resistance ability induced by HKDC1 overexpression in GC cells. Our results highlight a critical role of HKDC1 in promoting glycolysis, tumorigenesis, and EMT of GC cells via activating the NF‐κB pathway. In addition, HKDC1‐mediated drug resistance was associated with DNA damage repair, which further activated NF‐κB signaling. HKDC1 upregulation may be used as a potential indicator for choosing an effective chemotherapy regimen for GC patients undergoing chemotherapy.
HKDC1 upregulation predicts resistance to cisplatin, oxaliplatin and 5‐FU in patients with GC. HKDC1 upregulation may be used as a potential indicator for choosing an effective chemotherapy regimen for GC patients undergoing chemotherapy.
Six triterpenic acids were separated and purified from the ethyl acetate extractive fraction of ethanol extracts of Potentilla parvifolia FISCH. using a variety of chromatographic methods. The ...neuroprotective effects of these triterpenoids were investigated in the present study, in which the okadaic acid induced neurotoxicity in human neuroblastoma SH-SY5Y cells were used as an Alzheimer’s disease cell model in vitro. The cell model was established with all trans-retinoic acid (5 µmol/L, 4 d) and okadaic acid (40 nmol/L, 6 h) treatments to induce tau phosphorylation and synaptic atrophy. Subsequently, the neuroprotective effects of these triterpenic acids were evaluated in vitro by this cell model. Results from the Western blot and morphology analysis suggested that compounds 3–6 had the better neuroprotective effects. Furthermore, we tested the level of mitochondrial reactive oxygen species and mitochondrial membrane potential of these compounds in SH-SY5Y cells by flow cytometry technology to investigate the potential neuroprotective mechanism of these compounds. All of the results indicated that maybe the mechanism of compounds 5 and 6 is to protect the cell from mitochondrial oxidative stress injuries.
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