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Developmental Cell Apr 2024Control of protein stoichiometry is essential for cell function. Mitochondrial oxidative phosphorylation (OXPHOS) presents a complex stoichiometric challenge as the...
Control of protein stoichiometry is essential for cell function. Mitochondrial oxidative phosphorylation (OXPHOS) presents a complex stoichiometric challenge as the ratio of the electron transport chain (ETC) and ATP synthase must be tightly controlled, and assembly requires coordinated integration of proteins encoded in the nuclear and mitochondrial genome. How correct OXPHOS stoichiometry is achieved is unknown. We identify the Mitochondrial Regulatory hub for respiratory Assembly (MiRA) platform, which synchronizes ETC and ATP synthase biogenesis in yeast. Molecularly, this is achieved by a stop-and-go mechanism: the uncharacterized protein Mra1 stalls complex IV assembly. Two "Go" signals are required for assembly progression: binding of the complex IV assembly factor Rcf2 and Mra1 interaction with an Atp9-translating mitoribosome induce Mra1 degradation, allowing synchronized maturation of complex IV and the ATP synthase. Failure of the stop-and-go mechanism results in cell death. MiRA controls OXPHOS assembly, ensuring correct stoichiometry of protein machineries encoded by two different genomes.
Topics: Oxidative Phosphorylation; Saccharomyces cerevisiae; Mitochondria; Saccharomyces cerevisiae Proteins; Mitochondrial Proton-Translocating ATPases; Electron Transport Complex IV; Mitochondrial Proteins
PubMed: 38508182
DOI: 10.1016/j.devcel.2024.02.011 -
RNA (New York, N.Y.) Oct 2023Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in...
Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered amino-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of protein complexes according to their amino-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together, these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.
Topics: Humans; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; RecQ Helicases; Edetic Acid; DNA Damage; RNA; Ribonucleoproteins; Ribosomes
PubMed: 37460154
DOI: 10.1261/rna.079755.123 -
Nucleic Acids Research Apr 2024Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are...
Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.
Topics: R-Loop Structures; Saccharomyces cerevisiae Proteins; Transcription Termination, Genetic; Ribonuclease H; Saccharomyces cerevisiae; RNA Polymerase II; Transcription Factors; Transcription, Genetic; Exoribonucleases
PubMed: 38281203
DOI: 10.1093/nar/gkae033 -
Cell Reports Nov 2023RNA-binding proteins (RBPs) interact with mRNA to form supramolecular complexes called messenger ribonucleoprotein (mRNP) particles. These dynamic assemblies direct and...
RNA-binding proteins (RBPs) interact with mRNA to form supramolecular complexes called messenger ribonucleoprotein (mRNP) particles. These dynamic assemblies direct and regulate individual steps of gene expression; however, their composition and functional importance remain largely unknown. Here, we develop a total internal reflection fluorescence-based single-molecule imaging assay to investigate stoichiometry and co-occupancy of 15 RBPs within mRNPs from Saccharomyces cerevisiae. We show compositional heterogeneity of single mRNPs and plasticity across different growth conditions, with major co-occupants of mRNPs containing the nuclear cap-binding complex identified as Yra1 (1-10 copies), Nab2 (1-6 copies), and Npl3 (1-6 copies). Multicopy Yra1-bound mRNPs are specifically co-occupied by the THO complex and assembled on mRNAs biased by transcript length and RNA secondary structure. Yra1 depletion results in decreased compaction of nuclear mRNPs demonstrating a packaging function. Together, we provide a quantitative framework for gene- and condition-dependent RBP occupancy and stoichiometry in individual nuclear mRNPs.
Topics: Nuclear Proteins; Ribonucleoproteins; RNA, Messenger; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37963019
DOI: 10.1016/j.celrep.2023.113415 -
Scientific Reports Jan 2024During myocardial injury, inflammatory mediators and oxidative stress significantly increase to impair cardiac mitochondria. Emerging evidence has highlighted interplays...
During myocardial injury, inflammatory mediators and oxidative stress significantly increase to impair cardiac mitochondria. Emerging evidence has highlighted interplays between circadian protein-period 2 (Per2) and mitochondrial metabolism. However, besides circadian rhythm regulation, the direct role of Per2 in mitochondrial performance particularly following acute stress, remains unknown. In this study, we aim to determine the importance of Per2 protein's regulatory role in mitochondrial function following exposure to inflammatory cytokine TNFα and oxidative stressor HO in human cardiomyocytes. Global warm ischemia (37 °C) significantly impaired complex I activity with concurrently reduced mitochondrial Per2 in adult mouse hearts. TNFα or HO decreased Per2 protein levels and damaged mitochondrial respiratory function in adult mouse cardiomyocytes. Next, mitochondrial membrane potential ([Formula: see text] ) using JC-1 fluorescence probe and mitochondrial respiration capacity via Seahorse Cell Mito Stress Test were then detected in Per2 or control siRNA transfected AC16 Human Cardiomyocytes (HCM) that were subjected to 2 h-treatment of TNFα (100 ng/ml) or HO (100 μM). After 4 h-treatment, cell death was also measured using Annexin V and propidium iodide apoptosis kit through flow cytometry. We found that knockdown of Per2 enhanced TNFα-induced cell death and TNFα- or HO-disrupted [Formula: see text], as well as TNFα- or HO-impaired mitochondrial respiration function. In conclusion, Per2 knockdown increases likelihood of cell death and mitochondrial dysfunction in human cardiomyocytes exposed to either TNFα or HO, supporting the protective role of Per2 in HCM during stress with a focus on mitochondrial function.
Topics: Animals; Humans; Mice; Apoptosis; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocytes, Cardiac; Oxidative Stress; Period Circadian Proteins; Tumor Necrosis Factor-alpha
PubMed: 38221535
DOI: 10.1038/s41598-024-51799-w -
International Journal of Molecular... Nov 2023Membrane-spanning portions of proteins' polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of... (Review)
Review
Membrane-spanning portions of proteins' polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of TMDs from proteins of various families, be that receptors, ion channels, enzymes etc., have been under scrutiny on the part of the scientific community for the last few decades. The reason for such attention is that, apart from their obvious role as an "anchor" in ensuring the correct orientation of the protein's extra-membrane domains (in most cases functionally important), TMDs often actively and directly contribute to the operation of "the protein machine". They are capable of transmitting signals across the membrane, interacting with adjacent TMDs and membrane-proximal domains, as well as with various ligands, etc. Structural data on TMD arrangement are still fragmentary at best due to their complex molecular organisation as, most commonly, dynamic oligomers, as well as due to the challenges related to experimental studies thereof. Inter alia, this is especially true for viral fusion proteins, which have been the focus of numerous studies for quite some time, but have provoked unprecedented interest in view of the SARS-CoV-2 pandemic. However, despite numerous structure-centred studies of the spike (S) protein effectuating target cell entry in coronaviruses, structural data on the TMD as part of the entire spike protein are still incomplete, whereas this segment is known to be crucial to the spike's fusogenic activity. Therefore, in attempting to bring together currently available data on the structure and dynamics of spike proteins' TMDs, the present review aims to tackle a highly pertinent task and contribute to a better understanding of the molecular mechanisms underlying virus-mediated fusion, also offering a rationale for the design of novel efficacious methods for the treatment of infectious diseases caused by SARS-CoV-2 and related viruses.
Topics: Humans; Membrane Fusion; Protein Domains; Viral Fusion Proteins; Peptides; SARS-CoV-2
PubMed: 38003610
DOI: 10.3390/ijms242216421 -
PLoS Genetics Jan 2024Eukaryotic chromatin is organized into either silenced heterochromatin or relaxed euchromatin regions, which controls the accessibility of transcriptional machinery and...
Eukaryotic chromatin is organized into either silenced heterochromatin or relaxed euchromatin regions, which controls the accessibility of transcriptional machinery and thus regulates gene expression. In fission yeast, Schizosaccharomyces pombe, Set1 is the sole H3K4 methyltransferase and is mainly enriched at the promoters of actively transcribed genes. In contrast, Clr4 methyltransferase initiates H3K9 methylation, which has long been regarded as a hallmark of heterochromatic silencing. Lsd1 and Lsd2 are two highly conserved H3K4 and H3K9 demethylases. As these histone-modifying enzymes perform critical roles in maintaining histone methylation patterns and, consequently, gene expression profiles, cross-regulations among these enzymes are part of the complex regulatory networks. Thus, elucidating the mechanisms that govern their signaling and mutual regulations remains crucial. Here, we demonstrated that C-terminal truncation mutants, lsd1-ΔHMG and lsd2-ΔC, do not compromise the integrity of the Lsd1/2 complex but impair their chromatin-binding capacity at the promoter region of target genomic loci. We identified protein-protein interactions between Lsd1/2 and Raf2 or Swd2, which are the subunits of the Clr4 complex (CLRC) and Set1-associated complex (COMPASS), respectively. We showed that Clr4 and Set1 modulate the protein levels of Lsd1 and Lsd2 in opposite ways through the ubiquitin-proteasome-dependent pathway. During heat stress, the protein levels of Lsd1 and Lsd2 are upregulated in a Set1-dependent manner. The increase in protein levels is crucial for differential gene expression under stress conditions. Together, our results support a cross-regulatory model by which Set1 and Clr4 methyltransferases control the protein levels of Lsd1/2 demethylases to shape the dynamic chromatin landscape.
Topics: Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Histones; Histone-Lysine N-Methyltransferase; Cell Cycle Proteins; Heterochromatin; Transcription Factors
PubMed: 38181050
DOI: 10.1371/journal.pgen.1011107 -
Biochimica Et Biophysica Acta.... Jun 2024Iron‑sulfur (FeS) clusters are cofactors of numerous proteins involved in essential cellular functions including respiration, protein translation, DNA synthesis and... (Review)
Review
Iron‑sulfur (FeS) clusters are cofactors of numerous proteins involved in essential cellular functions including respiration, protein translation, DNA synthesis and repair, ribosome maturation, anti-viral responses, and isopropylmalate isomerase activity. Novel FeS proteins are still being discovered due to the widespread use of cryogenic electron microscopy (cryo-EM) and elegant genetic screens targeted at protein discovery. A complex sequence of biochemical reactions mediated by a conserved machinery controls biosynthesis of FeS clusters. In eukaryotes, a remarkable epistasis has been observed: the mitochondrial machinery, termed ISC (Iron-Sulfur Cluster), lies upstream of the cytoplasmic machinery, termed CIA (Cytoplasmic Iron‑sulfur protein Assembly). The basis for this arrangement is the production of a hitherto uncharacterized intermediate, termed X-S or (Fe-S), produced in mitochondria by the ISC machinery, exported by the mitochondrial ABC transporter Atm1 (ABCB7 in humans), and then utilized by the CIA machinery for the cytoplasmic/nuclear FeS cluster assembly. Genetic and biochemical findings supporting this sequence of events are herein presented. New structural views of the Atm1 transport phases are reviewed. The key compartmental roles of glutathione in cellular FeS cluster biogenesis are highlighted. Finally, data are presented showing that every one of the ten core components of the mitochondrial ISC machinery and Atm1, when mutated or depleted, displays similar phenotypes: mitochondrial and cytoplasmic FeS clusters are both rendered deficient, consistent with the epistasis noted above.
Topics: Mitochondria; Iron-Sulfur Proteins; Humans; Cytoplasm; Saccharomyces cerevisiae Proteins; ATP-Binding Cassette Transporters; Saccharomyces cerevisiae; Mitochondrial Proteins; Glutathione
PubMed: 38641180
DOI: 10.1016/j.bbamcr.2024.119733 -
International Journal of Molecular... Sep 2023The evolving history of BRCA1 research demonstrates the profound interconnectedness of a single protein within the web of crucial functions in human cells. Mutations in... (Review)
Review
The evolving history of BRCA1 research demonstrates the profound interconnectedness of a single protein within the web of crucial functions in human cells. Mutations in BRCA1, a tumor suppressor gene, have been linked to heightened breast and ovarian cancer risks. However, despite decades of extensive research, the mechanisms underlying BRCA1's contribution to tissue-specific tumor development remain elusive. Nevertheless, much of the BRCA1 protein's structure, function, and interactions has been elucidated. Individual regions of BRCA1 interact with numerous proteins to play roles in ubiquitination, transcription, cell checkpoints, and DNA damage repair. At a cellular scale, these BRCA1 functions coordinate tumor suppression, R-loop prevention, and cellular differentiation, all of which may contribute to BRCA1's role in cancer tissue specificity. As research on BRCA1 and breast cancer continues to evolve, it will become increasingly evident that modern materials such as Bisphenol A should be examined for their relationship with DNA stability, cancer incidence, and chemotherapy. Overall, this review offers a comprehensive understanding of BRCA1's many roles at a molecular, cellular, organismal, and environmental scale. We hope that the knowledge gathered here highlights both the necessity of BRCA1 research and the potential for novel strategies to prevent and treat cancer in individuals carrying BRCA1 mutations.
Topics: Humans; Female; BRCA1 Protein; Breast Neoplasms; Ovarian Neoplasms; Breast; DNA Repair
PubMed: 37762577
DOI: 10.3390/ijms241814276 -
BMC Bioinformatics Aug 2023Proteins often assemble into higher-order complexes to perform their biological functions. Such protein-protein interactions (PPI) are often experimentally measured for...
BACKGROUND
Proteins often assemble into higher-order complexes to perform their biological functions. Such protein-protein interactions (PPI) are often experimentally measured for pairs of proteins and summarized in a weighted PPI network, to which community detection algorithms can be applied to define the various higher-order protein complexes. Current methods include unsupervised and supervised approaches, often assuming that protein complexes manifest only as dense subgraphs. Utilizing supervised approaches, the focus is not on how to find them in a network, but only on learning which subgraphs correspond to complexes, currently solved using heuristics. However, learning to walk trajectories on a network to identify protein complexes leads naturally to a reinforcement learning (RL) approach, a strategy not extensively explored for community detection. Here, we develop and evaluate a reinforcement learning pipeline for community detection on weighted protein-protein interaction networks to detect new protein complexes. The algorithm is trained to calculate the value of different subgraphs encountered while walking on the network to reconstruct known complexes. A distributed prediction algorithm then scales the RL pipeline to search for novel protein complexes on large PPI networks.
RESULTS
The reinforcement learning pipeline is applied to a human PPI network consisting of 8k proteins and 60k PPI, which results in 1,157 protein complexes. The method demonstrated competitive accuracy with improved speed compared to previous algorithms. We highlight protein complexes such as C4orf19, C18orf21, and KIAA1522 which are currently minimally characterized. Additionally, the results suggest TMC04 be a putative additional subunit of the KICSTOR complex and confirm the involvement of C15orf41 in a higher-order complex with HIRA, CDAN1, ASF1A, and by 3D structural modeling.
CONCLUSIONS
Reinforcement learning offers several distinct advantages for community detection, including scalability and knowledge of the walk trajectories defining those communities. Applied to currently available human protein interaction networks, this method had comparable accuracy with other algorithms and notable savings in computational time, and in turn, led to clear predictions of protein function and interactions for several uncharacterized human proteins.
Topics: Humans; Protein Interaction Maps; Algorithms; Transcription Factors; Protein Interaction Mapping; Computational Biology; Glycoproteins; Nuclear Proteins; Cell Cycle Proteins; Molecular Chaperones
PubMed: 37532987
DOI: 10.1186/s12859-023-05425-7