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Scientific Reports Jun 2024Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the...
Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1) and cyclin F (CCNF). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1, providing multiplexed information at single-cell resolution. In addition to SOD1 and CCNF, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.
Topics: Amyotrophic Lateral Sclerosis; Proteostasis; Humans; Superoxide Dismutase-1; Protein Folding; Mutation; Cell Line; Mice; Animals
PubMed: 38879591
DOI: 10.1038/s41598-024-64366-0 -
Microbial Cell Factories Jun 2024Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their...
BACKGROUND
Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.
RESULTS
In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.
CONCLUSIONS
In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.
Topics: Promoter Regions, Genetic; Saccharomycetales; Bioreactors; Recombinant Proteins; Mixed Function Oxygenases; Methanol
PubMed: 38879507
DOI: 10.1186/s12934-024-02451-9 -
Scientific Reports Jun 2024The twin-arginine translocation (Tat) system transports folded proteins across energized biological membranes in bacteria, plastids, and plant mitochondria. In...
The twin-arginine translocation (Tat) system transports folded proteins across energized biological membranes in bacteria, plastids, and plant mitochondria. In Escherichia coli, the three membrane proteins TatA, TatB and TatC associate to enable Tat transport. While TatB and TatC together form complexes that bind Tat-dependently transported proteins, the TatA component is responsible for the permeabilization of the membrane during transport. With wild type Tat systems, the TatB- and TatC-containing Tat complexes TC1 and TC2 can be differentiated. Their TatA content has not been resolved, nor could they be assigned to any step of the translocation mechanism. It is therefore a key question of current Tat research to understand how TatA associates with Tat systems during transport. By analyzing affinity-purified Tat complexes with mutations in TatC that selectively enrich either TC1 or TC2, we now for the first time demonstrate that both Tat complexes associate with TatA, but the larger TC2 recruits significantly more TatA than the smaller TC1. Most TatA co-purified as multimeric clusters. Using site-specific photo cross-linking, we could detect TatA-TatC interactions only near TatC transmembrane helices 5 and 6. Substrate-binding did not change the interacting positions but affected the stability of the interaction, pointing to a substrate-induced conformational transition. Together, our findings indicate that TatA clusters associate with TatBC without being integrated into the complex by major rearrangements. The increased TatA affinity of the larger Tat complex TC2 suggests that functional assembly is advanced in this complex.
Topics: Escherichia coli Proteins; Escherichia coli; Membrane Transport Proteins; Cell Membrane; Protein Transport; Protein Folding; Protein Binding; Mutation
PubMed: 38877109
DOI: 10.1038/s41598-024-64547-x -
The Journal of Biological Chemistry Jun 2024The voltage-gated potassium ion channel K11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome...
The voltage-gated potassium ion channel K11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which pharmacological chaperones like E-4031 can rescue. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes of wild-type (WT) K11.1 or trafficking-deficient channel variants in the presence or absence of E4031. We identified 572 core K11.1 protein interactors. Trafficking-deficient variants K11.1-G601S and K11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We confirmed previous findings that the proteasome is critical for K11.1 degradation. Our report provides the first comprehensive characterization of protein quality control mechanisms of K11.1. We find extensive interactome remodeling associated with trafficking-deficient K11.1 variants, and with pharmacological chaperone rescue of K11.1 cell surface expression. The identified protein interactions could be targeted therapeutically to improve K11.1 trafficking and treat Long QT Syndrome.
PubMed: 38876300
DOI: 10.1016/j.jbc.2024.107465 -
Archives of Biochemistry and Biophysics Jun 2024Mutation of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (F508del CFTR) yields a protein unstable at physiological...
Mutation of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (F508del CFTR) yields a protein unstable at physiological temperatures that is rapidly degraded in the cell. This mutation is present in about 90% of cystic fibrosis patients, hence there is great interest in compounds reversing its instability. We have previously reported the expression of the mutated protein at low temperature and its purification in detergent. Here we describe the use of the protein to screen compounds present in a library of Federal Drug Administration (FDA) - approved drugs and also in a small natural product library. The kinetics of unfolding of F508del CFTR at 37°C were probed by the increase in solvent-exposed cysteine residues accessible to a fluorescent reporter molecule. This occurred in a bi-exponential manner with a major (≈60%) component of half-life around 5 minutes and a minor component of around 60 minutes. The faster kinetics match those observed for loss of channel activity of F508del CFTR in cells at 37°C. Most compounds tested had no effect on the fluorescence increase, but some were identified that significantly slowed the kinetics. The general properties of these compounds, and any likely mechanisms for inducing stability in purified CFTR are discussed. These experimental data may be useful for artificial intelligence - aided design of CFTR-specific drugs and in the identification of stabilizing additives for membrane proteins (in general).
PubMed: 38876247
DOI: 10.1016/j.abb.2024.110050 -
Chemical Science Jun 2024An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome...
An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome (CCHS). Recent studies demonstrated that the alanine tract expansion influences PHOX2B folding and activity. Therefore, structural information on PHOX2B is an important target for obtaining clues to elucidate the insurgence of the alanine expansion-related syndrome and also for defining a viable therapy. Here we report by NMR spectroscopy the structural characterization of the homeodomain (HD) of PHOX2B and HD + C-terminus PHOX2B protein, free and in the presence of the target DNA. The obtained structural data are then exploited to obtain a structural model of the PHOX2B-DNA interaction. In addition, the variant +7Ala, responsible for one of the most frequent forms of the syndrome, was analysed, showing different conformational proprieties in solution and a strong propensity to aggregation. Our data suggest that the elongated poly-alanine tract would be related to disease onset through a loss-of-function mechanism. Overall, this study paves the way for the future rational design of therapeutic drugs, suggesting as a possible therapeutic route the use of specific anti-aggregating molecules capable of preventing variant aggregation and possibly restoring the DNA-binding activity of PHOX2B.
PubMed: 38873078
DOI: 10.1039/d3sc06427a -
Frontiers in Veterinary Science 2024-glycosylation is a highly conserved glycan modification that plays crucial roles in various physiological processes, including protein folding, trafficking, and signal...
-glycosylation is a highly conserved glycan modification that plays crucial roles in various physiological processes, including protein folding, trafficking, and signal transduction. Porcine deltacoronavirus (PDCoV) poses a newly emerging threat to the global porcine industry. The spike protein of PDCoV exhibits a high level of -glycosylation; however, its role in viral infection remains poorly understood. In this study, we applied a lentivirus-based entry reporter system to investigate the role of -glycosylation on the viral spike protein during PDCoV entry stage. Our findings demonstrate that -glycosylation at positions 652 and 661 of the viral spike protein significantly reduces the infectivity of PDCoV pseudotyped virus. Overall, our results unveil a novel function of -glycosylation in PDCoV infection, highlighting its potential for facilitating the development of antiviral strategies.
PubMed: 38872801
DOI: 10.3389/fvets.2024.1430113 -
Journal of Molecular Biology Jun 2024TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to...
TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.
PubMed: 38871177
DOI: 10.1016/j.jmb.2024.168652 -
IScience Jun 2024Multiple metabolic events occur in mitochondria. Mitochondrial protein translocation from the cytoplasm across compartments depends on the amino acid sequence within the...
Multiple metabolic events occur in mitochondria. Mitochondrial protein translocation from the cytoplasm across compartments depends on the amino acid sequence within the precursor. At the mitochondria associated-ER membrane, misfolding of a mitochondrial targeted protein prior to import ablates metabolism. CYP11A1, cytochrome P450 cholesterol side chain cleavage enzyme (SCC), is imported from the cytoplasm to mitochondrial matrix catalyzing cholesterol to pregnenolone, an essential step for metabolic processes and mammalian survival. Multiple steps regulate the availability of an actively folded SCC; however, the mechanism is unknown. We identified that a dry molten globule state of SCC exists in the matrix by capturing intermediate protein folding steps dictated by its C-terminus. The intermediate dry molten globule state in the mitochondrial matrix of living cells is stable with a limited network of interaction and is inactive. The dry molten globule is activated with hydrogen ions availability, triggering cleavage of cholesterol sidechain, and initiating steroidogenesis.
PubMed: 38868187
DOI: 10.1016/j.isci.2024.110039 -
FEBS Open Bio Jun 2024The mitochondrial intermembrane space hosts a machinery for oxidative protein folding, the mitochondrial disulfide relay. This machinery imports a large number of... (Review)
Review
The mitochondrial intermembrane space hosts a machinery for oxidative protein folding, the mitochondrial disulfide relay. This machinery imports a large number of soluble proteins into the compartment, where they are retained through oxidative folding. Additionally, the disulfide relay enhances the stability of many proteins by forming disulfide bonds. In this review, we describe the mitochondrial disulfide relay in human cells, its components, and their coordinated collaboration in mechanistic detail. We also discuss the human pathologies associated with defects in this machinery and its protein substrates, providing a comprehensive overview of its biological importance and implications for health.
PubMed: 38867508
DOI: 10.1002/2211-5463.13839