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Protein Expression and Purification Mar 2024Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements,...
Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements, bacteriophage protection, stress response and possibly persister formation. A major bottleneck in the study of TA toxins is the production of sufficient amounts of well-folded, functional protein. Here we examine alternative approaches for obtaining the VcParE2 toxin from Vibrio cholerae. VcParE2 can be successfully produced via bacterial expression in presence of its cognate antitoxin VcParD2, followed by on-column unfolding and refolding. Alternatively, the toxin can be expressed in Spodoptera frugiperda (Sf9) insect cells. The latter requires disruption of the VcparE2 gene via introduction of an insect cell intron. Both methods provide protein with similar structural and functional characteristics.
Topics: Bacterial Toxins; Antitoxins; Vibrio cholerae; Operon; Bacterial Proteins
PubMed: 37977515
DOI: 10.1016/j.pep.2023.106403 -
PLoS Computational Biology Nov 2023The possibility of the protein backbone adopting lasso-like entangled motifs has attracted increasing attention. After discovering the surprising abundance of natively...
The possibility of the protein backbone adopting lasso-like entangled motifs has attracted increasing attention. After discovering the surprising abundance of natively entangled protein domain structures, it was shown that misfolded entangled subpopulations might become thermosensitive or escape the homeostasis network just after translation. To investigate the role of entanglement in shaping folding kinetics, we introduce a novel indicator and analyze simulations of a coarse-grained, structure-based model for two small single-domain proteins. The model recapitulates the well-known two-state folding mechanism of a non-entangled SH3 domain. However, despite its small size, a natively entangled antifreeze RD1 protein displays a rich refolding behavior, populating two distinct kinetic intermediates: a short-lived, entangled, near-unfolded state and a longer-lived, non-entangled, near-native state. The former directs refolding along a fast pathway, whereas the latter is a kinetic trap, consistently with known experimental evidence of two different characteristic times. Upon trapping, the natively entangled loop folds without being threaded by the N-terminal residues. After trapping, the native entangled structure emerges by either backtracking to the unfolded state or threading through the already formed but not yet entangled loop. Along the fast pathway, trapping does not occur because the native contacts at the closure of the lasso-like loop fold after those involved in the N-terminal thread, confirming previous predictions. Despite this, entanglement may appear already in unfolded configurations. Remarkably, a longer-lived, near-native intermediate, with non-native entanglement properties, recalls what was observed in cotranslational folding.
Topics: Protein Folding; Proteins; Physics; Kinetics; Protein Conformation
PubMed: 37956216
DOI: 10.1371/journal.pcbi.1011107 -
Journal of Lipid Research Dec 2023Protein aggregates arise naturally under normal physiological conditions, but their formation is accelerated by age or stress-induced protein misfolding. When the...
Protein aggregates arise naturally under normal physiological conditions, but their formation is accelerated by age or stress-induced protein misfolding. When the stressful event dissolves, these aggregates are removed by mechanisms, such as aggrephagy, chaperone-mediated autophagy, refolding attempts, or the proteasome. It was recently shown that mitochondria in yeast cells may support these primarily cytosolic processes. Protein aggregates attach to mitochondria, and misfolded proteins are transported into the matrix and degraded by mitochondria-specific proteases. Using a proximity labeling method and colocalization with an established stress granule (SG) marker, we were able to show that these mitochondria-localized aggregates that harbor the "super aggregator" Ola1p are, in fact, SGs. Our in vivo and in vitro studies have revealed that Ola1p can be transferred from mitochondria to lipid droplets (LDs). This "mitochondria to LD" aggregate transfer dampens proteotoxic effects. The LD-based protein aggregate removal system gains importance when other proteolytic systems fail. Furthermore, we were able to show that the distribution of SGs is drastically altered in LD-deficient yeast cells, demonstrating that LDs play a role in the SG life cycle.
Topics: Lipid Droplets; Mitochondria; Proteasome Endopeptidase Complex; Protein Aggregates; Saccharomyces cerevisiae; Stress Granules
PubMed: 37949369
DOI: 10.1016/j.jlr.2023.100473 -
Experimental & Molecular Medicine Nov 2023Osteoarthritis (OA) is a full-joint, multifactorial, degenerative and inflammatory disease that seriously affects the quality of life of patients due to its disabling...
Osteoarthritis (OA) is a full-joint, multifactorial, degenerative and inflammatory disease that seriously affects the quality of life of patients due to its disabling and pain-causing properties. ER stress has been reported to be closely related to the progression of OA. The inositol-requiring enzyme 1α/X-box-binding protein-1 spliced (IRE1α/XBP1s) pathway, which is highly expressed in the chondrocytes of OA patients, promotes the degradation and refolding of abnormal proteins during ER stress and maintains the stability of the ER environment of chondrocytes, but its function and the underlying mechanisms of how it contributes to the progression of OA remain unclear. This study investigates the role of IRE1α/ERN1 in OA. Specific deficiency of ERN1 in chondrocytes spontaneously resulted in OA-like cartilage destruction and accelerated OA progression in a surgically induced arthritis model. Local delivery of AdERN1 relieved degradation of the cartilage matrix and prevented OA development in an ACLT-mediated model. Mechanistically, progranulin (PGRN), an intracellular chaperone, binds to IRE1α, promoting its phosphorylation and splicing of XBP1u to generate XBP1s. XBP1s protects articular cartilage through TNF-α/ERK1/2 signaling and further maintains collagen homeostasis by regulating type II collagen expression. The chondroprotective effect of IRE1α/ERN1 is dependent on PGRN and XBP1s splicing. ERN1 deficiency accelerated cartilage degeneration in OA by reducing PGRN expression and XBP1s splicing, subsequently decreasing collagen II expression and triggering collagen structural abnormalities and an imbalance in collagen homeostasis. This study provides new insights into OA pathogenesis and the UPR and suggests that IRE1α/ERN1 may serve as a potential target for the treatment of joint degenerative diseases, including OA.
Topics: Humans; Protein Serine-Threonine Kinases; Progranulins; Endoribonucleases; Quality of Life; Osteoarthritis; Chondrocytes; Cartilage, Articular; Collagen; Homeostasis; X-Box Binding Protein 1
PubMed: 37907740
DOI: 10.1038/s12276-023-01106-w -
BioRxiv : the Preprint Server For... Oct 2023Mechanical force controls the opening and closing of mechanosensitive ion channels atop the hair bundles of the inner ear. The filamentous tip link connecting...
Mechanical force controls the opening and closing of mechanosensitive ion channels atop the hair bundles of the inner ear. The filamentous tip link connecting transduction channels to the tallest neighboring stereocilium modulates the force transmitted to the channels and thus changes their probability of opening. Each tip link comprises four molecules: a dimer of protocadherin 15 and a dimer of cadherin 23, all of which are stabilized by Ca binding. Using a high-speed optical trap to examine dimeric PCDH15, we find that the protein's configuration is sensitive to Ca and that the molecule exhibits limited unfolding at a physiological Ca concentration. PCDH15 can therefore modulate its stiffness without undergoing large unfolding events in physiological Ca conditions. The experimentally determined stiffness of PCDH15 accords with published values for the stiffness of the gating spring, the mechanical element that controls the opening of mechanotransduction channels. When PCDH15 has a point mutation, V507D, associated with non-syndromic hearing loss, unfolding events occur more frequently under tension and refolding events occur less often than in the wild-type protein. Our results suggest that the maintenance of appropriate tension in the gating spring is critical to the appropriate transmission of force to transduction channels, and hence to hearing.
PubMed: 37905108
DOI: 10.1101/2023.10.17.562769 -
Vaccines Sep 2023Influenza virus infections represent an ongoing public health threat as well as an economic burden. Although seasonal influenza vaccines have been available for some...
Influenza virus infections represent an ongoing public health threat as well as an economic burden. Although seasonal influenza vaccines have been available for some decades, efforts are being made to generate new efficient, flexible, and cost-effective technologies to be transferred into production. Our work describes the development of a model influenza hemagglutinin antigen that is capable of inducing protection against viral challenge in mice. High amounts of the H1 hemagglutinin ectodomain, HA, were expressed in a bacterial system as insoluble inclusion bodies. Solubilization was followed by a thorough differential scanning fluorimetry (DSF)-guided optimization of refolding, which allows for fast and reliable screening of several refolding conditions, yielding tens of milligrams/L of folded protein. Structural and functional analysis revealed native-like folding as well as the presence of a mix of monomers and oligomers in solution. Mice immunized with HA were protected when exposed to influenza A virus as opposed to mice that received full-length denatured protein. Sera of mice immunized with HA showed both high titers of antigen-specific IgG1 and IgG2a isotypes as well as viral neutralization activity. These results prove the feasibility of the recombinant bacterial expression system coupled with DSF-guided refolding in providing influenza hemagglutinin for vaccine development.
PubMed: 37896924
DOI: 10.3390/vaccines11101520 -
International Journal of Molecular... Oct 2023Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing...
Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In , fibrillar IbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue IbpB induces the transition of IbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic possesses only a single sHSP, IbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of IbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the multi-chaperone system is driven with the ability of IbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to IbpA and IbpB, IbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of IbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to IbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, IbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.
Topics: Heat-Shock Proteins; Acholeplasma laidlawii; Escherichia coli Proteins; HSP70 Heat-Shock Proteins; Molecular Chaperones; Escherichia coli; Heat-Shock Proteins, Small
PubMed: 37895124
DOI: 10.3390/ijms242015445 -
Veterinary World Sep 2023abortion in mares is caused by subspecies serovar infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are...
BACKGROUND AND AIM
abortion in mares is caused by subspecies serovar infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of abortion in mares
MATERIALS AND METHODS
Outer membrane protein X from the antigen was synthesized and expressed in using the pET28 vector. Transformed cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA.
RESULTS
After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from . High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares.
CONCLUSION
Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses.
PubMed: 37859952
DOI: 10.14202/vetworld.2023.1790-1795 -
Protein Expression and Purification Feb 2024Antibody-mimetic drug conjugate (AMDC) is a cancer cell-targeted drug delivery system based on the non-covalent binding of mutated streptavidin and modified biotin,...
Antibody-mimetic drug conjugate (AMDC) is a cancer cell-targeted drug delivery system based on the non-covalent binding of mutated streptavidin and modified biotin, namely Cupid and Psyche. However, the development of AMDCs is hampered by difficulties in post-translational modification or poor internalization activity. Here, we report an expression, refolding, and purification method for AMDC using a variable heavy chain of heavy chain-only antibodies (VHHs). Monomeric anti-HER2 VHH fused to Cupid was expressed in Escherichia coli inclusion bodies. Solubilization and refolding at optimized reducing conditions and pH levels were selected to form a functional, tetrameric protein (anti-HER2 VHH-Cupid) that can be easily purified based on molecular weight. Anti-HER2 VHH-Cupid non-covalently creates a tight complex with Psyche linked to a potent DNA-alkylating agent, duocarmycin. This complex can be absorbed by the HER2-expressing human breast cancer cell line, KPL-4, and kills KPL-4 cells in vitro and in vivo. The production of a targeting protein with internalizing activity, combined with the non-covalent conjugation of a highly potent payload, renders AMDC a promising platform for developing cancer-targeted therapy.
Topics: Humans; Duocarmycins; Immunoconjugates; Receptor, ErbB-2; Cell Line, Tumor; Drug Delivery Systems
PubMed: 37797818
DOI: 10.1016/j.pep.2023.106375 -
Nature Dec 2023Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory...
Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion. Spike opening exposes domain S1, allowing it to bind to proteinaceous receptors, and is also thought to enable protein refolding during membrane fusion. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1. This binding triggers the transition of S1 domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.
Topics: Humans; Allosteric Regulation; Betacoronavirus; Common Cold; Cryoelectron Microscopy; Molecular Dynamics Simulation; Polysaccharides; Protein Binding; Protein Conformation; Sialic Acids; Spike Glycoprotein, Coronavirus; Immune Evasion
PubMed: 37794193
DOI: 10.1038/s41586-023-06599-z