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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 -
Nature Communications Apr 2024Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or...
Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.
Topics: DNA Damage; Heat-Shock Proteins, Small; Intranuclear Inclusion Bodies; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sumoylation; Vesicular Transport Proteins
PubMed: 38615096
DOI: 10.1038/s41467-024-47615-8 -
Materials Horizons Nov 2023Regulating protein folding including assisting folding, preventing misfolding and aggregation, and facilitating refolding of proteins are of significant importance for...
Regulating protein folding including assisting folding, preventing misfolding and aggregation, and facilitating refolding of proteins are of significant importance for retaining protein's biological activities. Here, we report a mixed shell polymeric micelle (MSPM)-based self-cooperative nanochaperone (self--nChap) with enhanced activity to facilitate protein refolding. This self--nChap was fabricated by introducing Hsp40-mimetic artificial carriers into the traditional nanochaperone to cooperate with the Hsp70-mimetic confined hydrophobic microdomains. The artificial carrier facilitates transfer and immobilization of client proteins into confined hydrophobic microdomains, by which significantly improving self--nChap's capability to inhibit unfolding and aggregation of client proteins, and finally facilitating refolding. Compared to traditional nanochaperones, the self--nChap significantly enhances the thermal stability of horseradish peroxidase (HRP) epicyclically under harsher conditions. Moreover, the self--nChap efficiently protects misfolding-prone proteins, such as immunoglobulin G (IgG) antibody from thermal denaturation, which is hardly achieved using traditional nanochaperones. In addition, a kinetic partitioning mechanism was devised to explain how self--nChap facilitates refolding by regulating the cooperative effect of kinetics between the nanochaperone and client proteins. This work provides a novel strategy for the design of protein folding regulatory materials, including nanochaperones.
Topics: Humans; Protein Refolding; HSP70 Heat-Shock Proteins; Polymers
PubMed: 37843027
DOI: 10.1039/d3mh00619k -
The Journal of Physical Chemistry. B Aug 2023Ionic liquids (ILs) are known to stabilize protein conformations in aqueous medium. Importantly, ILs can also act as refolding additives in urea-driven denaturation of...
Ionic liquids (ILs) are known to stabilize protein conformations in aqueous medium. Importantly, ILs can also act as refolding additives in urea-driven denaturation of proteins. However, despite the importance of the problem, detailed microscopic understanding of the counteraction effects of ILs on urea-induced protein denaturation remains elusive. In this work, atomistic molecular dynamics (MD) simulations of the protein α-lactalbumin have been carried out in pure aqueous medium, in 8 M binary urea-water solution and in ternary urea-IL-water solutions containing ammonium-based ethyl ammonium acetate (EAA) as the IL at different concentrations (1-4 M). Attempts have been made to quantify detailed molecular-level understanding of the origin behind the counteraction effects of the IL on urea-induced partial unfolding of the protein. The calculations revealed significant conformational changes of the protein with multiple free energy minima due to its partial unfolding in binary urea-water solution. The counteraction effect of the IL was evident from the enhanced structural rigidity of the protein with propensity to transform into a single native free energy minimum state in ternary urea-IL-water solutions. Such an effect has been found to be associated with preferential direct binding of the IL components with the protein and simultaneous expulsion of urea from the interface, thereby providing additional stabilization of the protein in ternary solutions. Most importantly, modified rearrangement of the hydrogen bond network at the interface due to the formation of stronger protein-cation (PC) and protein-anion (PA) hydrogen bonds by breaking relatively weaker protein-urea (PU) and protein-water (PW) hydrogen bonds has been recognized as the microscopic origin behind the counteraction effects of EAA on urea-induced partial unfolding of the protein.
Topics: Ionic Liquids; Lactalbumin; Peptides; Ammonium Compounds; Urea; Molecular Dynamics Simulation; Water; Transcription Factors; Protein Denaturation
PubMed: 37574910
DOI: 10.1021/acs.jpcb.3c03223 -
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 -
International Journal of Biological... Nov 2023This study investigated effects of carboxymethylcellulose sodium (CMC) on the conformational evolution of pea protein during the high moisture extrusion process. The...
Changes in physicochemical and structural properties of pea protein during the high moisture extrusion process: Effects of carboxymethylcellulose sodium and different extrusion zones.
This study investigated effects of carboxymethylcellulose sodium (CMC) on the conformational evolution of pea protein during the high moisture extrusion process. The morphological observation showed that the addition of CMC facilitated the formation of fibrous structure of pea protein. In comparison with the pea protein in the melting zone and extrudate, the combination of CMC increased the denaturation enthalpy of pea protein by 2.09 % and 2.34 %. Compared with the material in the mixing zone, the degree of grafting between CMC and pea protein in the die was enhanced by 98.95 %. In general, the supplementation of CMC depressed the exposure of hydrophobic groups in the pea protein. In the extrusion barrel, the CMC increased the unfolding of protein molecular chains while it promoted the refolding of protein chains in the die. For the extrudate, the addition of CMC decreased the contents of α-helix and β-sheet of pea protein by 9.67 % and 6.93 % while the contents of β-turn and random coil were increased, leading to changes in the molecular weight distribution of protein molecules. In conclusion, these results provided new strategies toward producing the high-quality pea protein-based meat analogues by adding CMC.
Topics: Carboxymethylcellulose Sodium; Pea Proteins; Pisum sativum; Water; Chemical Phenomena; Hydrophobic and Hydrophilic Interactions; Molecular Weight; Protein Conformation; Thermodynamics
PubMed: 37591439
DOI: 10.1016/j.ijbiomac.2023.126350 -
World Journal of Microbiology &... May 2024Nanobodies are the smallest known antigen-binding molecules to date. Their small size, good tissue penetration, high stability and solubility, ease of expression,... (Review)
Review
Nanobodies are the smallest known antigen-binding molecules to date. Their small size, good tissue penetration, high stability and solubility, ease of expression, refolding ability, and negligible immunogenicity in the human body have granted them excellence over conventional antibodies. Those exceptional attributes of nanobodies make them promising candidates for various applications in biotechnology, medicine, protein engineering, structural biology, food, and agriculture. This review presents an overview of their structure, development methods, advantages, possible challenges, and applications with special emphasis on infectious diseases-related ones. A showcase of how nanobodies can be harnessed for applications including neutralization of viruses and combating antibiotic-resistant bacteria is detailed. Overall, the impact of nanobodies in vaccine design, rapid diagnostics, and targeted therapies, besides exploring their role in deciphering microbial structures and virulence mechanisms are highlighted. Indeed, nanobodies are reshaping the future of infectious disease prevention and treatment.
Topics: Single-Domain Antibodies; Humans; Communicable Diseases; Animals; Biotechnology; Protein Engineering
PubMed: 38771414
DOI: 10.1007/s11274-024-03990-4 -
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 -
Biochemical and Biophysical Research... Jun 2024Human heavy-chain ferritin is a naturally occurring protein with high stability and multifunctionality in biological systems. This study aims to utilize a prokaryotic...
Human heavy-chain ferritin is a naturally occurring protein with high stability and multifunctionality in biological systems. This study aims to utilize a prokaryotic expression system to produce recombinant human heavy-chain ferritin nanoparticles and investigate their targeting ability in brain tissue. The human heavy-chain ferritin gene was cloned into the prokaryotic expression vector pET28a and transformed into Escherichia coli BL21 (DE3) competent cells to explore optimal expression conditions. The recombinant protein was then purified to evaluate its immunoreactivity and characteristics. Additionally, the distribution of the administered protein in normal mice and its permeability in an in vitro blood-brain barrier (BBB) model were measured. The results demonstrate that the purified protein can self-assemble extracellularly into nano-cage structures of approximately 10 nm and is recognized by corresponding antibodies. The protein effectively penetrates the blood-brain barrier and exhibits slow clearance in mouse brain tissue, showing excellent permeability in the in vitro BBB model. This study highlights the stable expression of recombinant human heavy-chain ferritin using the Escherichia coli prokaryotic expression system, characterized by favorable nano-cage structures and biological activity. Its exceptional brain tissue targeting and slow metabolism lay an experimental foundation for its application in neuropharmaceutical delivery and vaccine development fields.
Topics: Animals; Humans; Recombinant Proteins; Mice; Blood-Brain Barrier; Brain; Escherichia coli; Nanoparticles; Ferritins; Apoferritins; Tissue Distribution
PubMed: 38640729
DOI: 10.1016/j.bbrc.2024.149939 -
Phytomedicine : International Journal... Jan 2024Liver cancer is one of common types of cancer with poor prognosis and limited therapies. Heat shock proteins (HSP) are molecular chaperones that have important roles in...
BACKGROUND
Liver cancer is one of common types of cancer with poor prognosis and limited therapies. Heat shock proteins (HSP) are molecular chaperones that have important roles in tumorigenesis, and emerging as therapeutic targets. Artemisinin and rhein are natural agents from Artemisia annua L. and Rheum undulatum L., respectively. Both rhein and artemisinin have anticancer effects; however, the molecular targets of rhein remain to be identified. It is also unclear whether rhein can synergize with artemisinin derivatives to inhibit liver cancer.
PURPOSE
We aim to identify the targets of rhein in the treatment of hepatocarcinoma and determine the effects of combining rhein and artemisinin derivatives on liver cancer cells.
METHODS
The targets of rhein were detected by mass spectrometry and validated by rhein-proteins interaction assays. The effects of rhein on the chaperone activity of HSP72/HSC70/GRP78 were determined by luciferase refolding assays. Cell viability and apoptosis were determined by CCK8 and flow cytometry assays. For in vivo study, xenograft tumor models were established and treated with rhein and artesunate. Tumor growth was monitored regularly.
RESULTS
Mass spectrometry analysis of rhein-binding proteins in HepG2 cells revealed that HSP72, HSC70 and GRP78 were more profoundly pulled down by rhein-crosslinked sepharose 4B beads compared to the control beads. Further experiments demonstrated that rhein directly interacted with HSP72/HSC70/GRP78 proteins, and inhibit their activity of refolding denatured luciferase. Meanwhile, rhein induced proteasomal degradation of HIF1α and β-catenin. Artesunate or dihydroartemisinin in combination with knockdown of both HSP72 and HSC70 significantly inhibited cell viability. The HSP70/HSC70/GRP78 inhibitors VER-155,008 and rhein phenocopied HSP72/HSC70 knockdown, synergizing with artesunate or dihydroartemisinin to inhibit hepatocarcinoma cell viability. Combinatorial treatment with rhein and artemisinin derivatives significantly induced hepatocarcinoma cell apoptosis, and inhibited tumor growth in vivo.
CONCLUSIONS
The current study demonstrates that rhein is a novel HSP72/HSC70/GRP78 inhibitor that suppresses the chaperone activity of HSP70s. Dual inhibition of HSP72 and HSC70 can enhance the sensitivity of hepatocarcinoma cells to artemisinin derivatives. Combined treatment with artemisinin derivative and rhein significantly inhibits hepatocarcinoma. Artemisinin derivatives in combination with dual inhibition of HSP72 and HSC70 represents a new approach to improve cancer therapy.
Topics: Humans; Endoplasmic Reticulum Chaperone BiP; HSC70 Heat-Shock Proteins; Artesunate; HSP70 Heat-Shock Proteins; Artemisinins; Molecular Chaperones; Liver Neoplasms; Luciferases; HSP72 Heat-Shock Proteins
PubMed: 37897861
DOI: 10.1016/j.phymed.2023.155156