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Life Science Alliance Aug 2024Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron...
Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.
Topics: Ribosomes; Membrane Proteins; Endoplasmic Reticulum; Protein Biosynthesis; Cryoelectron Microscopy; SEC Translocation Channels; Molecular Chaperones; Protein Transport; Models, Molecular
PubMed: 38866426
DOI: 10.26508/lsa.202302496 -
Science Advances Jun 2024The accumulation of protein aggregates is a hallmark of many diseases, including Alzheimer's disease. As a major pillar of the proteostasis network, autophagy mediates...
The accumulation of protein aggregates is a hallmark of many diseases, including Alzheimer's disease. As a major pillar of the proteostasis network, autophagy mediates the degradation of protein aggregates. The autophagy cargo receptor p62 recognizes ubiquitin on proteins and cooperates with TAX1BP1 to recruit the autophagy machinery. Paradoxically, protein aggregates are not degraded in various diseases despite p62 association. Here, we reconstituted the recognition by the autophagy receptors of physiological and pathological Tau forms. Monomeric Tau recruits p62 and TAX1BP1 via the sequential actions of the chaperone and ubiquitylation machineries. In contrast, Tau fibrils from Alzheimer's disease brains are recognized by p62 but fail to recruit TAX1BP1. This failure is due to the masking of fibrils ubiquitin moieties by p62. Tau fibrils are resistant to deubiquitylation, and, thus, this nonproductive interaction of p62 with the fibrils is irreversible. Our results shed light on the mechanism underlying autophagy evasion by protein aggregates and their consequent accumulation in disease.
Topics: Autophagy; Humans; tau Proteins; Ubiquitination; Sequestosome-1 Protein; Alzheimer Disease; Protein Binding; Protein Aggregates; Intracellular Signaling Peptides and Proteins; Ubiquitin; Neoplasm Proteins
PubMed: 38865459
DOI: 10.1126/sciadv.adm8449 -
Cell Reports Jun 2024The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5' untranslated region (UTR) of the fragile X messenger...
The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene contains polymorphic CGG repeats, the length of which has differing effects on FMR1 expression and human health, including the neurodevelopmental disorder fragile X syndrome. We deleted the CGG repeats in the FMR1 gene (0CGG) in human stem cells and examined the effects on differentiated neurons. 0CGG neurons have altered subcellular localization of FMR1 mRNA and protein, and differential expression of cellular stress proteins compared with neurons with normal repeats (31CGG). In addition, 0CGG neurons have altered responses to glucocorticoid receptor (GR) activation, including FMR1 mRNA localization, GR chaperone HSP90α expression, GR localization, and cellular stress protein levels. Therefore, the CGG repeats in the FMR1 gene are important for the homeostatic responses of neurons to stress signals.
Topics: Humans; Fragile X Mental Retardation Protein; Neurons; RNA, Messenger; Receptors, Glucocorticoid; Stress, Physiological; 5' Untranslated Regions; HSP90 Heat-Shock Proteins; Trinucleotide Repeats; Trinucleotide Repeat Expansion
PubMed: 38865241
DOI: 10.1016/j.celrep.2024.114330 -
Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain.Protein Science : a Publication of the... Jul 2024Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone...
Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.
Topics: Molecular Chaperones; Binding Sites; Humans; Protein Domains; Adaptor Proteins, Signal Transducing; Models, Molecular; Hydrophobic and Hydrophilic Interactions
PubMed: 38864729
DOI: 10.1002/pro.5063 -
Frontiers in Immunology 2024Spinal Muscular Atrophy (SMA), a neurodegenerative disorder, extends its impact beyond the nervous system. The central protein implicated in SMA, Survival Motor Neuron... (Review)
Review
Spinal Muscular Atrophy (SMA), a neurodegenerative disorder, extends its impact beyond the nervous system. The central protein implicated in SMA, Survival Motor Neuron (SMN) protein, is ubiquitously expressed and functions in fundamental processes such as alternative splicing, translation, cytoskeletal dynamics and signaling. These processes are relevant for all cellular systems, including cells of the immune system such as macrophages. Macrophages are capable of modulating their splicing, cytoskeleton and expression profile in order to fulfil their role in tissue homeostasis and defense. However, less is known about impairment or dysfunction of macrophages lacking SMN and the subsequent impact on the immune system of SMA patients. We aimed to review the potential overlaps between SMN functions and macrophage mechanisms highlighting the need for future research, as well as the current state of research addressing the role of macrophages in SMA.
Topics: Humans; Macrophages; Muscular Atrophy, Spinal; Animals; Survival of Motor Neuron 1 Protein; Signal Transduction
PubMed: 38863697
DOI: 10.3389/fimmu.2024.1375428 -
Scientific Reports Jun 2024At present, liquid storage is the most efficient method for pig semen preservation. This approach relies upon reducing sperm metabolism, allowing for the maintenance of...
At present, liquid storage is the most efficient method for pig semen preservation. This approach relies upon reducing sperm metabolism, allowing for the maintenance of cell lifespan. In this context, the study of proteins that could protect sperm during liquid storage is of high relevance. The 70 kDa Heat Shock Protein (HSP70) is an anti-apoptotic protein that has been reported to be relevant to sperm survival. Thus, we explored the role of HSP70 during prolonged storage of pig semen at 17 °C. Six semen pools were incubated with YM-1 (0, 0.05, 0.1 and 0.2 μM), an HSP70 inhibitor, and stored at 17 °C for 21 days. On days 0, 4, 10, 14 and 21, sperm quality and function were evaluated through flow cytometry and Computer-Assisted Sperm Analysis (CASA), and HSP70 activity and chromatin condensation were also determined. While inhibition of HSP70 increased progressive motility, Ca and Reactive Oxygen Species (ROS) levels, and mitochondrial activity during the first 10 days of storage, it had a detrimental effect on sperm motility after 14 and 21 days. In spite of this, sperm viability was not altered. We can conclude that HSP70 contributes to the liquid storage of pig semen because it keeps mitochondrial activity low, which is needed for the maintenance of sperm function.
Topics: Animals; Male; HSP70 Heat-Shock Proteins; Spermatozoa; Semen Preservation; Swine; Sperm Motility; Reactive Oxygen Species; Mitochondria; Semen Analysis; Cell Survival; Calcium
PubMed: 38862610
DOI: 10.1038/s41598-024-64488-5 -
Nature Communications Jun 2024Metabolic remodeling is a strategy for tumor survival under stress. However, the molecular mechanisms during the metabolic remodeling of colorectal cancer (CRC) remain...
Metabolic remodeling is a strategy for tumor survival under stress. However, the molecular mechanisms during the metabolic remodeling of colorectal cancer (CRC) remain unclear. Melanocyte proliferating gene 1 (MYG1) is a 3'-5' RNA exonuclease and plays a key role in mitochondrial functions. Here, we uncover that MYG1 expression is upregulated in CRC progression and highly expressed MYG1 promotes glycolysis and CRC progression independent of its exonuclease activity. Mechanistically, nuclear MYG1 recruits HSP90/GSK3β complex to promote PKM2 phosphorylation, increasing its stability. PKM2 transcriptionally activates MYC and promotes MYC-medicated glycolysis. Conversely, c-Myc also transcriptionally upregulates MYG1, driving the progression of CRC. Meanwhile, mitochondrial MYG1 on the one hand inhibits oxidative phosphorylation (OXPHOS), and on the other hand blocks the release of Cyt c from mitochondria and inhibits cell apoptosis. Clinically, patients with KRAS mutation show high expression of MYG1, indicating a high level of glycolysis and a poor prognosis. Targeting MYG1 may disturb metabolic balance of CRC and serve as a potential target for the diagnosis and treatment of CRC.
Topics: Animals; Female; Humans; Male; Mice; Apoptosis; Carrier Proteins; Cell Line, Tumor; Cell Nucleus; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Glycolysis; HSP90 Heat-Shock Proteins; Membrane Proteins; Mice, Nude; Mitochondria; Oxidative Phosphorylation; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Thyroid Hormone-Binding Proteins; Thyroid Hormones
PubMed: 38862489
DOI: 10.1038/s41467-024-49221-0 -
Neuromolecular Medicine Jun 2024Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease affecting motor neurons. Pathological forms of Tar-DNA binding protein-43 (TDP-43), involving...
Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease affecting motor neurons. Pathological forms of Tar-DNA binding protein-43 (TDP-43), involving its mislocalisation to the cytoplasm and the formation of misfolded inclusions, are present in almost all ALS cases (97%), and ~ 50% cases of the related condition, frontotemporal dementia (FTD), highlighting its importance in neurodegeneration. Previous studies have shown that endoplasmic reticulum protein 57 (ERp57), a member of the protein disulphide isomerase (PDI) family of redox chaperones, is protective against ALS-linked mutant superoxide dismutase (SOD1) in neuronal cells and transgenic SOD1 mouse models. However, it remains unclear whether ERp57 is protective against pathological TDP-43 in ALS. Here, we demonstrate that ERp57 is protective against key features of TDP-43 pathology in neuronal cells. ERp57 inhibited the mislocalisation of TDP-43 from the nucleus to the cytoplasm. In addition, ERp57 inhibited the number of inclusions formed by ALS-associated variant TDP-43 and reduced the size of these inclusions. ERp57 was also protective against ER stress and induction of apoptosis. Furthermore, ERp57 modulated the steady-state expression levels of TDP-43. This study therefore demonstrates a novel mechanism of action of ERp57 in ALS. It also implies that ERp57 may have potential as a novel therapeutic target to prevent the TDP-43 pathology associated with neurodegeneration.
Topics: Protein Disulfide-Isomerases; Amyotrophic Lateral Sclerosis; DNA-Binding Proteins; Humans; Inclusion Bodies; Animals; Mice; Cell Nucleus; Cytoplasm; Neurons; Superoxide Dismutase-1; Mutation
PubMed: 38861223
DOI: 10.1007/s12017-024-08787-0 -
Archives of Microbiology Jun 2024Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be...
Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be a beneficial adaptation to the cold environment. Here we show that Cpn60 from the psychrophile Pseudoalteromonas haloplanktis (Ph) maintains its double-ring structure also in the cold. A strongly reduced ATPase activity keeps the chaperonin in an energy-saving dormant state, until binding of client protein activates it. Ph Cpn60 in complex with co-chaperonin Ph Cpn10 efficiently assists in protein folding up to 55 °C. Moreover, we show that recombinant expression of Ph Cpn60 can provide its host Escherichia coli with improved viability under low temperature growth conditions. These properties of the Ph chaperonin may make it a valuable tool in the folding and stabilization of psychrophilic proteins.
Topics: Pseudoalteromonas; Cold Temperature; Protein Folding; Bacterial Proteins; Escherichia coli; Chaperonin 60; Recombinant Proteins; Chaperonins; Adenosine Triphosphatases
PubMed: 38861015
DOI: 10.1007/s00203-024-04019-y -
Journal of Advanced Research Jun 2024Salmonella Enteritidis has brought great harm to public health, animal production and food safety worldwide. The biofilm formed by Salmonella Enteritidis plays a...
INTRODUCTION
Salmonella Enteritidis has brought great harm to public health, animal production and food safety worldwide. The biofilm formed by Salmonella Enteritidis plays a critical role in microbial cross-contamination. Small non-coding RNAs (sRNAs) have been demonstrated to be responsible for regulating the formation of biofilm. The sRNA SaaS has been identified previously, that promotes pathogenicity by regulating invasion and virulence factors. However, whether the SaaS is implicated in regulating biofilm formation in abiotic surfaces remains unclear.
OBJECTIVES
This study aimed to clarify the effect of SaaS in Salmonella Enteritidis and explore the modulatory mechanism on the biofilm formation.
METHODS
Motility characteristics and total biomass of biofilm of test strains were investigated by the phenotypes in three soft agar plates and crystal violet staining in polystyrene microplates. Studies of microscopic structure and extracellular polymeric substances (EPS) of biofilm on solid surfaces were carried out using confocal laser scanning microscope (CLSM) and Raman spectra. Transcriptomics and proteomics were applied to analyze the changes of gene expression and EPS component. The RNA-protein pull-down and promoter-reporter β-galactosidase activity assays were employed to analyze RNA binding proteins and identify target mRNAs, respectively.
RESULTS
SaaS inhibits biofilm formation by repressing the adhesion potential and the secretion of EPS components. Integration of transcriptomics and proteomics analysis revealed that SaaS strengthened the expression of the flagellar synthesis system and downregulated the expression of curli amyloid fibers. Furthermore, RNA-protein pull-down interactome datasets indicated that SaaS binds to Hfq (an RNA molecular chaperone protein, known as a host factor for phage Qbeta RNA replication) uniquely among 193 candidate proteins, and promoter-reporter β-galactosidase activity assay confirmed target mRNAs including hilD, cheA, and csgA.
CONCLUSION
SaaS inhibits the properties of bacterial mobility, perturbs the secretion of EPS, and contributes to the inhibition of biofilm formation by interacting with target mRNA (hilD, cheA, and csgA) through the Hfq-mediated pathway.
PubMed: 38852803
DOI: 10.1016/j.jare.2024.06.008