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The Journal of Biological Chemistry Sep 2023Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits...
Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous β-subunits. To better reveal β-subunit functions, we engineered a human cell line devoid of endogenous Na β-subunits and their immediate phylogenetic relatives. This new cell line, β-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the β-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac Na α-subunit, Na1.5, was highly sensitive to β-subunit modulation and revealed that each β-subunit and even MPZ imparted unique gating properties. Furthermore, combining β1 and β2 with Na1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of β-subunits to regulate Na1.5 activity and can be used to improve the characterization of other α/β Na complexes.
Topics: Humans; Action Potentials; Cell Line; Intracellular Signaling Peptides and Proteins; NAV1.5 Voltage-Gated Sodium Channel; Phosphoproteins; Protein Subunits; Voltage-Gated Sodium Channel beta Subunits; Mutation
PubMed: 37544648
DOI: 10.1016/j.jbc.2023.105132 -
The Journal of Biological Chemistry Mar 2024RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is... (Review)
Review
RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.
Topics: Coenzymes; Endoribonucleases; Evolution, Molecular; Protein Subunits; Ribonuclease P; RNA Processing, Post-Transcriptional; RNA, Catalytic; RNA, Transfer; Substrate Specificity; Eukaryota
PubMed: 38336296
DOI: 10.1016/j.jbc.2024.105729 -
Mathematical Biosciences Apr 2023We consider a general mathematical model for protein subunit vaccine with a focus on the MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2, and use the...
We consider a general mathematical model for protein subunit vaccine with a focus on the MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2, and use the model to study immunological outcomes in the humoral and cell-mediated arms of the immune response from vaccination. The mathematical model is fit to vaccine clinical trial data. We elucidate the role of Interferon-γ and Interleukin-4 in stimulating the immune response of the host. Model results, and results from a sensitivity analysis, show that a balance between the T1 and T2 arms of the immune response is struck, with the T1 response being dominant. The model predicts that two-doses of the vaccine at 28 days apart will result in approximately 85% humoral immunity loss relative to peak immunity approximately 6 months post dose 1.
Topics: Humans; COVID-19 Vaccines; Protein Subunits; COVID-19; SARS-CoV-2; Interferon-gamma; Vaccination; Antibodies, Viral
PubMed: 36773843
DOI: 10.1016/j.mbs.2023.108970 -
Nature Communications Jun 2022The activity of V-ATPase is well-known to be regulated by reversible dissociation of its V and V domains in response to growth factor stimulation, nutrient sensing, and...
The activity of V-ATPase is well-known to be regulated by reversible dissociation of its V and V domains in response to growth factor stimulation, nutrient sensing, and cellular differentiation. The molecular basis of its regulation by an endogenous modulator without affecting V-ATPase assembly remains unclear. Here, we discover that a lysosome-anchored protein termed (mammalian Enhancer-of-Akt-1-7 (mEAK7)) binds to intact V-ATPase. We determine cryo-EM structure of human mEAK7 in complex with human V-ATPase in native lipid-containing nanodiscs. The structure reveals that the TLDc domain of mEAK7 engages with subunits A, B, and E, while its C-terminal domain binds to subunit D, presumably blocking V-V torque transmission. Our functional studies suggest that mEAK7, which may act as a V-ATPase inhibitor, does not affect the activity of V-ATPase in vitro. However, overexpression of mEAK7 in HCT116 cells that stably express subunit a4 of V-ATPase represses the phosphorylation of ribosomal protein S6. Thus, this finding suggests that mEAK7 potentially links mTOR signaling with V-ATPase activity.
Topics: Humans; Protein Subunits; Vacuolar Proton-Translocating ATPases
PubMed: 35672408
DOI: 10.1038/s41467-022-30899-z -
RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits.Molecular Cell Apr 2023RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important...
RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3' end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3' end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells.
Topics: Animals; RNA Polymerase II; Transcription, Genetic; Ribosomal Proteins; Ribosomes; Protein Subunits; Mammals
PubMed: 36924766
DOI: 10.1016/j.molcel.2023.02.028 -
Cells Apr 2021The MutL family of DNA mismatch repair proteins (MMR) acts to maintain genomic integrity in somatic and meiotic cells. In baker's yeast, the MutL homolog (MLH) MMR... (Review)
Review
The MutL family of DNA mismatch repair proteins (MMR) acts to maintain genomic integrity in somatic and meiotic cells. In baker's yeast, the MutL homolog (MLH) MMR proteins form three heterodimeric complexes, MLH1-PMS1, MLH1-MLH2, and MLH1-MLH3. The recent discovery of human PMS2 (homolog of baker's yeast PMS1) and MLH3 acting independently of human MLH1 in the repair of somatic double-strand breaks questions the assumption that MLH1 is an obligate subunit for MLH function. Here we provide a summary of the canonical roles for MLH factors in DNA genomic maintenance and in meiotic crossover. We then present the phenotypes of cells lacking specific MLH subunits, particularly in meiotic recombination, and based on this analysis, propose a model for an independent early role for MLH3 in meiosis to promote the accurate segregation of homologous chromosomes in the meiosis I division.
Topics: Animals; DNA; DNA Repair; Homologous Recombination; Humans; Meiosis; MutL Proteins; Protein Subunits
PubMed: 33923939
DOI: 10.3390/cells10040948 -
Cell Jul 2020Excitatory neurotransmission meditated by glutamate receptors including N-methyl-D-aspartate receptors (NMDARs) is pivotal to brain development and function. NMDARs are...
Excitatory neurotransmission meditated by glutamate receptors including N-methyl-D-aspartate receptors (NMDARs) is pivotal to brain development and function. NMDARs are heterotetramers composed of GluN1 and GluN2 subunits, which bind glycine and glutamate, respectively, to activate their ion channels. Despite importance in brain physiology, the precise mechanisms by which activation and inhibition occur via subunit-specific binding of agonists and antagonists remain largely unknown. Here, we show the detailed patterns of conformational changes and inter-subunit and -domain reorientation leading to agonist-gating and subunit-dependent competitive inhibition by providing multiple structures in distinct ligand states at 4 Å or better. The structures reveal that activation and competitive inhibition by both GluN1 and GluN2 antagonists occur by controlling the tension of the linker between the ligand-binding domain and the transmembrane ion channel of the GluN2 subunit. Our results provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition, which are fundamental to the brain physiology.
Topics: Binding Sites; Binding, Competitive; Cryoelectron Microscopy; Crystallography, X-Ray; Dimerization; Glutamic Acid; Glycine; Humans; Ligands; Molecular Dynamics Simulation; Protein Structure, Quaternary; Protein Subunits; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins
PubMed: 32610085
DOI: 10.1016/j.cell.2020.05.052 -
Current Opinion in Structural Biology Oct 2019Electron cryo-microscopy (cryoEM) is used to determine structures of biological molecules, including multi-protein complexes. Maps at better than 3.0Å resolution are... (Review)
Review
Electron cryo-microscopy (cryoEM) is used to determine structures of biological molecules, including multi-protein complexes. Maps at better than 3.0Å resolution are relatively straightforward to interpret since atomic models of proteins and nucleic acids can be built directly. Still, these resolutions are often difficult to achieve, and map quality frequently varies within a structure. This results in data that are challenging to interpret, especially when crystal structures or suitable homology models are not available. Recent advances in mass spectrometry techniques, computational methods and model building tools facilitate subunit/domain fitting into maps, elucidation of protein contacts, and de novo generation of atomic models. Here, we review techniques for map interpretation and provide examples from recent studies of multi-protein complexes.
Topics: Cryoelectron Microscopy; Mass Spectrometry; Models, Molecular; Protein Subunits; Proteins; Signal-To-Noise Ratio
PubMed: 31362190
DOI: 10.1016/j.sbi.2019.06.009 -
Frontiers in Immunology 2022Under the background of the severe human health and world economic burden caused by COVID-19, the attenuation of vaccine protection efficacy, and the prevalence and...
Under the background of the severe human health and world economic burden caused by COVID-19, the attenuation of vaccine protection efficacy, and the prevalence and immune escape of emerging variants of concern (VOCs), the third dose of booster immunization has been put on the agenda. Systems biology approaches can help us gain new perspectives on the characterization of immune responses and the identification of factors underlying vaccine-induced immune efficacy. We analyzed the antibody signature and transcriptional responses of participants vaccinated with COVID-19 inactivated vaccine and protein subunit vaccine as a third booster dose. The results from the antibody indicated that the third booster dose was effective, and that heterologous vaccination with the protein subunit vaccine as a booster dose induced stronger humoral immune responses than the homologous vaccination with inactivated vaccine, and might be more effective against VOCs. In transcriptomic analysis, protein subunit vaccine induced more differentially expressed genes that were significantly associated with many important innate immune pathways. Both the homologous and heterologous boosters could increase the effectiveness against COVID-19, and compared with the inactivated vaccine, the protein subunit vaccine, mediated a stronger humoral immune response and had a more significant correlation with the innate immune function module, which provided certain data support for the third booster immunization strategy.
Topics: Humans; Immunity, Humoral; Transcriptome; Protein Subunits; Immunization, Secondary; COVID-19; Vaccines, Inactivated; Vaccines, Subunit
PubMed: 36341453
DOI: 10.3389/fimmu.2022.1027180 -
Biological Chemistry Oct 2020The mitochondrial ATP synthase is a multi-subunit enzyme complex located in the inner mitochondrial membrane which is essential for oxidative phosphorylation under... (Review)
Review
The mitochondrial ATP synthase is a multi-subunit enzyme complex located in the inner mitochondrial membrane which is essential for oxidative phosphorylation under physiological conditions. In this review, we analyse the enzyme functions involved in cancer progression by dissecting specific conditions in which ATP synthase contributes to cancer development or metastasis. Moreover, we propose the role of ATP synthase in the formation of the permeability transition pore (PTP) as an additional mechanism which controls tumour cell death. We further describe transcriptional and translational modifications of the enzyme subunits and of the inhibitor protein IF1 that may promote adaptations leading to cancer metabolism. Finally, we outline ATP synthase gene mutations and epigenetic modifications associated with cancer development or drug resistance, with the aim of highlighting this enzyme complex as a potential novel target for future anti-cancer therapy.
Topics: Animals; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Mitochondrial Permeability Transition Pore; Mitochondrial Proton-Translocating ATPases; Mutation; Neoplasms; Protein Modification, Translational; Protein Subunits; Proteins; ATPase Inhibitory Protein
PubMed: 32769215
DOI: 10.1515/hsz-2020-0157