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Autorepression of yeast Hsp70 cochaperones by intramolecular interactions involving their J-domains.Cell Stress & Chaperones Apr 2024The 70 kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s...
The 70 kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s to trigger ATP hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1's ATPase, indicate the presence of yet uncharacterized intramolecular dynamic interactions between the J-domains and the remaining C-terminal segments of these proteins. Taken together, the data suggest an autoregulatory role to these intramolecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol.
Topics: Saccharomyces cerevisiae; HSP40 Heat-Shock Proteins; Saccharomyces cerevisiae Proteins; Protein Binding; HSP70 Heat-Shock Proteins; Molecular Chaperones; Adenosine Triphosphatases; Adenosine Triphosphate
PubMed: 38521349
DOI: 10.1016/j.cstres.2024.03.008 -
Biomarker Research Mar 2024Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) originate from preleukemic hematopoietic conditions, such as clonal hematopoiesis of indeterminate... (Review)
Review
Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) originate from preleukemic hematopoietic conditions, such as clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenia of undetermined significance (CCUS) and have variable outcomes despite the successful implementation of targeted therapies. The prognosis differs depending on the molecular subgroup. In patients with TP53 mutations, the most inferior outcomes across independent studies were observed. Myeloid malignancies with TP53 mutations have complex cytogenetics and extensive structural variants. These factors contribute to worse responses to induction therapy, demethylating agents, or venetoclax-based treatments. Survival of patients with biallelic TP53 gene mutations is often less than one year but this depends on the type of treatment applied. It is still controversial whether the allelic state of mutant TP53 impacts the outcomes in patients with AML and high-risk MDS. Further studies are needed to justify estimating TP53 LOH status for better risk assessment. Yet, TP53-mutated MDS, MDS/AML and AML are now classified separately in the International Consensus Classification (ICC). In the clinical setting, the wild-type p53 protein is reactivated pharmacologically by targeting p53/MDM2/MDM4 interactions and mutant p53 reactivation is achieved by refolding the DNA binding domain to wild-type-like conformation or via targeted degradation of the mutated protein. This review discusses our current understanding of p53 biology in MDS and AML and the promises and failures of wild-type and mutant p53 reactivation in the clinical trial setting.
PubMed: 38481290
DOI: 10.1186/s40364-024-00579-9 -
BMC Plant Biology Mar 2024Primary response genes play a pivotal role in translating short-lived stress signals into sustained adaptive responses. In this study, we investigated the involvement of...
BACKGROUND
Primary response genes play a pivotal role in translating short-lived stress signals into sustained adaptive responses. In this study, we investigated the involvement of ATL80, an E3 ubiquitin ligase, in the dynamics of gene expression following water deprivation stress. We observed that ATL80 is rapidly activated within minutes of water deprivation stress perception, reaching peak expression around 60 min before gradually declining. ATL80, despite its post-translational regulation role, emerged as a key player in modulating early gene expression responses to water deprivation stress.
RESULTS
The impact of ATL80 on gene expression was assessed using a time-course microarray analysis (0, 15, 30, 60, and 120 min), revealing a burst of differentially expressed genes, many of which were associated with various stress responses. In addition, the diversity of early modulation of gene expression in response to water deprivation stress was significantly abolished in the atl80 mutant compared to wild-type plants. A subset of 73 genes that exhibited a similar expression pattern to ATL80 was identified. Among them, several are linked to stress responses, including ERF/AP2 and WRKY transcription factors, calcium signaling genes, MAP kinases, and signaling peptides. Promoter analysis predicts enrichment of binding sites for CAMTA1 and CAMTA5, which are known regulators of rapid stress responses. Furthermore, we have identified a group of differentially expressed ERF/AP2 transcription factors, proteins associated with folding and refolding, as well as pinpointed core module genes which are known to play roles in retrograde signaling pathways that cross-referenced with the early ATL80 transcriptome.
CONCLUSIONS
Based on these findings, we propose that ATL80 may target one or more components within the retrograde signaling pathways for degradation. In essence, ATL80 serves as a bridge connecting these signaling pathways and effectively functions as an alarm signal.
Topics: Ubiquitin-Protein Ligases; Water Deprivation; Transcription Factors; Dehydration; Gene Expression; Gene Expression Regulation, Plant; Plant Proteins
PubMed: 38459432
DOI: 10.1186/s12870-024-04872-5 -
Chemical Reviews Mar 2024Proteins are highly labile molecules, thus requiring the presence of appropriate solvents and excipients in their liquid milieu to keep their stability and biological... (Review)
Review
Proteins are highly labile molecules, thus requiring the presence of appropriate solvents and excipients in their liquid milieu to keep their stability and biological activity. In this field, ionic liquids (ILs) have gained momentum in the past years, with a relevant number of works reporting their successful use to dissolve, stabilize, extract, and purify proteins. Different approaches in protein-IL systems have been reported, namely, proteins dissolved in () neat ILs, () ILs as co-solvents, () ILs as adjuvants, () ILs as surfactants, () ILs as phase-forming components of aqueous biphasic systems, and () IL-polymer-protein/peptide conjugates. Herein, we critically analyze the works published to date and provide a comprehensive understanding of the IL-protein interactions affecting the stability, conformational alteration, unfolding, misfolding, and refolding of proteins while providing directions for future studies in view of imminent applications. Overall, it has been found that the stability or purification of proteins by ILs is bispecific and depends on the structure of both the IL and the protein. The most promising IL-protein systems are identified, which is valuable when foreseeing market applications of ILs, e.g., in "protein packaging" and "detergent applications". Future directions and other possibilities of IL-protein systems in light-harvesting and biotechnology/biomedical applications are discussed.
Topics: Ionic Liquids; Proteins; Solvents; Water; Polymers
PubMed: 38437627
DOI: 10.1021/acs.chemrev.3c00551 -
ACS Bio & Med Chem Au Feb 2024Many cell-surface receptors are promising targets for chemical synthesis because of their critical roles in disease development. This synthetic approach enables...
Deciphering the Synthetic and Refolding Strategy of a Cysteine-Rich Domain in the Tumor Necrosis Factor Receptor (TNF-R) for Racemic Crystallography Analysis and d-Peptide Ligand Discovery.
Many cell-surface receptors are promising targets for chemical synthesis because of their critical roles in disease development. This synthetic approach enables investigations by racemic protein crystallography and ligand discovery by mirror-image methodologies. However, due to their complex nature, the chemical synthesis of a receptor can be a significant challenge. Here, we describe the chemical synthesis and folding of a central, cysteine-rich domain of the cell-surface receptor tumor necrosis factor 1 which is integral to binding of the cytokine TNF-α, namely, TNFR-1 CRD2. Racemic protein crystallography at 1.4 Å confirmed that the native binding conformation was preserved, and TNFR-1 CRD2 maintained its capacity to bind to TNF-α ( ≈ 7 nM). Encouraged by this discovery, we carried out mirror-image phage display using the enantiomeric receptor mimic and identified a d-peptide ligand for TNFR-1 CRD2 ( = 1 μM). This work demonstrated that cysteine-rich domains, including the central domains, can be chemically synthesized and used as mimics for investigations.
PubMed: 38404743
DOI: 10.1021/acsbiomedchemau.3c00060 -
Molecular Biology Reports Feb 2024SLC38A2 is a ubiquitously expressed Na-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II...
BACKGROUND
SLC38A2 is a ubiquitously expressed Na-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II diabetes and cancer. Despite its relevance in human physio-pathology, structure/function relationship studies and identification of ligands with regulatory roles are still in infancy.
METHODS AND RESULTS
The cDNA coding for SLC38A2 was cloned in the pET-28-Mistic vector, and the BL21 codon plus RIL strain was transformed with the recombinant construct. 0.5% glucose and oxygen availability were crucial for protein expression. The over-expressed hSNAT2-Mistic chimera was cleaved on column and purified by nickel-chelating affinity chromatography, with a yield of about 60 mg/Liter cell culture. The purified hSNAT2 was reconstituted in proteoliposomes in an active form with a right-side-out orientation with respect to the native membrane.
CONCLUSIONS
The addition of a Mistic tag at the N-terminus of the SNAT2 protein was crucial for its over-expression and purification. The purified protein was functionally active, representing a powerful tool for performing structure/function studies and testing ligands as inhibitors and/or activators.
Topics: Humans; Amino Acid Transport System A; Membrane Transport Proteins
PubMed: 38393484
DOI: 10.1007/s11033-023-08976-3 -
Microbial Cell Factories Feb 2024The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive...
BACKGROUND
The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive three-finger proteins is quite difficult.
RESULTS
We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant α-Βungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, α-Cobratoxin, MTα, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography.
CONCLUSIONS
Our pipeline allows refolding and purifying recombinant three finger proteins under optimized conditions and can be scaled up for massive production of three finger proteins. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones and should facilitate related research and applications.
Topics: Escherichia coli; Recombinant Proteins; Inclusion Bodies; Disulfides
PubMed: 38347541
DOI: 10.1186/s12934-024-02316-1 -
Cancers Feb 2024Heat shock proteins (HSPs) are developmentally conserved families of protein found in both prokaryotic and eukaryotic organisms. HSPs are engaged in a diverse range of... (Review)
Review
Heat shock proteins (HSPs) are developmentally conserved families of protein found in both prokaryotic and eukaryotic organisms. HSPs are engaged in a diverse range of physiological processes, including molecular chaperone activity to assist the initial protein folding or promote the unfolding and refolding of misfolded intermediates to acquire the normal or native conformation and its translocation and prevent protein aggregation as well as in immunity, apoptosis, and autophagy. These molecular chaperonins are classified into various families according to their molecular size or weight, encompassing small HSPs (e.g., HSP10 and HSP27), HSP40, HSP60, HSP70, HSP90, and the category of large HSPs that include HSP100 and ClpB proteins. The overexpression of HSPs is induced to counteract cell stress at elevated levels in a variety of solid tumors, including anticancer chemotherapy, and is closely related to a worse prognosis and therapeutic resistance to cancer cells. HSPs are also involved in anti-apoptotic properties and are associated with processes of cancer progression and development, such as metastasis, invasion, and cell proliferation. This review outlines the previously mentioned HSPs and their significant involvement in diverse mechanisms of tumor advancement and metastasis, as well as their contribution to identifying potential targets for therapeutic interventions.
PubMed: 38339390
DOI: 10.3390/cancers16030638 -
Journal of Molecular Biology Jul 2024The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the... (Review)
Review
The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the folding of newly synthesized proteins to the disassembly and refolding of protein aggregates. This multifunctionality of Hsp70 is governed by J-domain proteins (JDPs), which act as indispensable co-chaperones that target specific substrates to Hsp70. The number of distinct JDPs present in a species always outnumbers Hsp70, documenting JDP function in functional diversification of Hsp70. In this review, we describe the physiological roles of JDPs in the Saccharomyces cerevisiae PQC system, with a focus on the abundant JDP generalists, Zuo1, Ydj1 and Sis1, which function in fundamental cellular processes. Ribosome-bound Zuo1 cooperates with the Hsp70 chaperones Ssb1/2 in folding and assembly of nascent polypeptides. Ydj1 and Sis1 cooperate with the Hsp70 members Ssa1 to Ssa4 to exert overlapping functions in protein folding and targeting of newly synthesized proteins to organelles including mitochondria and facilitating the degradation of aberrant proteins by E3 ligases. Furthermore, they act in protein disaggregation reactions, though Ydj1 and Sis1 differ in their modes of Hsp70 cooperation and substrate specificities. This results in functional specialization as seen in prion propagation and the underlying dominant role of Sis1 in targeting Hsp70 for shearing of prion amyloid fibrils.
Topics: Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; HSP70 Heat-Shock Proteins; Protein Folding; HSP40 Heat-Shock Proteins; Molecular Chaperones; Protein Domains; Heat-Shock Proteins
PubMed: 38331212
DOI: 10.1016/j.jmb.2024.168484 -
ACS Omega Jan 2024protein refolding is one of the critical unit operations in manufacturing recombinant peptides expressed using as host cells. This study is focused on designing size...
protein refolding is one of the critical unit operations in manufacturing recombinant peptides expressed using as host cells. This study is focused on designing size exclusion chromatography-assisted refolding process for biosimilar recombinant parathyroid hormone. Inclusion bodies (IBs) of recombinant parathyroid hormone were solubilized at higher pH, and refolding was performed using size exclusion chromatography. In the first part of the investigation, DoE-based empirical optimization was performed to achieve a higher refolding yield for a biosimilar recombinant parathyroid hormone. The effect of solubilized inclusion body (IB) feed volume, concentration of IBs, and residence time on refolding of recombinant teriparatide was studied using the Box-Behnken design. Size exclusion chromatography (SEC)-assisted refolding was performed at 8 °C at pH 10.5 by using 20 mM Tris buffer. The maximum refolding yield of 98.12% was achieved at feed volume (12.5% of CV) and 20 mg/mL inclusion body (IB) concentration with a residence time of 50 min and a purity of 66.1% based on densitometric analysis using SDS-PAGE. In the latter part of the investigation, the general rate mechanistic model framework for size exclusion chromatography was developed and validated with the experimental results. The developed model helped in the accurate prediction of the elution volumes and product yield. The developed model also helps to predict the elution performance of a scalable column a priori. Post refolding, the formation of the native peptide structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structures. The developed hybrid process development approach is a valuable tool toachieve high-yield, scalable refolding conditions for recombinant proteins without disulfide bonds.
PubMed: 38284095
DOI: 10.1021/acsomega.3c04463