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Nature Dec 2020Recent developments in high-throughput reverse genetics have revolutionized our ability to map gene function and interactions. The power of these approaches depends on...
Recent developments in high-throughput reverse genetics have revolutionized our ability to map gene function and interactions. The power of these approaches depends on their ability to identify functionally associated genes, which elicit similar phenotypic changes across several perturbations (chemical, environmental or genetic) when knocked out. However, owing to the large number of perturbations, these approaches have been limited to growth or morphological readouts. Here we use a high-content biochemical readout, thermal proteome profiling, to measure the proteome-wide protein abundance and thermal stability in response to 121 genetic perturbations in Escherichia coli. We show that thermal stability, and therefore the state and interactions of essential proteins, is commonly modulated, raising the possibility of studying a protein group that is particularly inaccessible to genetics. We find that functionally associated proteins have coordinated changes in abundance and thermal stability across perturbations, owing to their co-regulation and physical interactions (with proteins, metabolites or cofactors). Finally, we provide mechanistic insights into previously determined growth phenotypes that go beyond the deleted gene. These data represent a rich resource for inferring protein functions and interactions.
Topics: Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Mutant Proteins; Mutation; Phenotype; Protein Stability; Proteome; Proteomics; Reverse Genetics; Temperature
PubMed: 33299184
DOI: 10.1038/s41586-020-3002-5 -
Biochimie Jan 2021NhaA antiporters are secondary integral membrane protein critical for maintaining the Na+/H+ cell homeostasis, as a result, they regulate fundamental processes like cell... (Review)
Review
NhaA antiporters are secondary integral membrane protein critical for maintaining the Na+/H+ cell homeostasis, as a result, they regulate fundamental processes like cell volume and intracellular pH. Exploration of the structural and functional properties can assist to make them effective human drug targets and mechanisms of salt-resistance in plants. NhaA proteins are integrated into cytoplasmic and intracellular membranes, transport 2H+/Na + across the membrane by the canonical alternating access mechanism. There are mutagenesis studies have done on Ec-NhaA predicting residues crucial for function and structure. The unique NhaA structural fold is formed in the middle of the membrane by two transmembrane segments (TMs), TM IV and XI which cross each other creating a delicate electrostatically balanced environment for the binding of Na+/H+. Previously, Asp164, Asp163 and Asp133 residues have been proposed as crucial for Na+/Li + binding on the based on crystal structure and mutation-based studies. However, the pathway and the binding sites for the two protons are still elusive and debatable. This review will provide comprehensive details on various mutations constructed in Ec-NhaA by different research groups using site-directed or random mutagenesis techniques. The selected residues for mutations are located on the sites which are more suspected to have a crucial role in function and structure on NhaA. This information on the single platform would accelerate further studies on the structure-function relationship on NhaA as well as will facilitate to predict the role of Na+/H+ antiporters in human diseases.
Topics: Binding Sites; Escherichia coli Proteins; Ions; Mutant Proteins; Mutation; Protein Conformation; Protein Domains; Sodium-Hydrogen Exchangers
PubMed: 33129932
DOI: 10.1016/j.biochi.2020.10.017 -
ACS Synthetic Biology Mar 2022In addition to its biological function, the stability of a protein is a major determinant for its applicability. Unfortunately, engineering proteins for improved... (Review)
Review
In addition to its biological function, the stability of a protein is a major determinant for its applicability. Unfortunately, engineering proteins for improved functionality usually results in destabilization of the protein. This so-called stability-function trade-off can be explained by the simple fact that the generation of a novel protein function─or the improvement of an existing one─necessitates the insertion of mutations, , deviations from the evolutionarily optimized wild-type sequence. In fact, it was demonstrated that gain-of-function mutations are not more destabilizing than other random mutations. The stability-function trade-off is a universal phenomenon during protein evolution that has been observed with completely different types of proteins, including enzymes, antibodies, and engineered binding scaffolds. In this review, we discuss three types of strategies that have been successfully deployed to overcome this omnipresent obstacle in protein engineering approaches: (i) using highly stable parental proteins, (ii) minimizing the extent of destabilization during functional engineering (by library optimization and/or coselection for stability and function), and (iii) repairing damaged mutants through stability engineering. The implementation of these strategies in protein engineering campaigns will facilitate the efficient generation of protein variants that are not only functional but also stable and therefore better-suited for subsequent applications.
Topics: Gene Library; Mutant Proteins; Mutation; Protein Engineering; Proteins
PubMed: 35258287
DOI: 10.1021/acssynbio.1c00512 -
Proceedings of the National Academy of... Aug 2022Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by...
Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant gene expression. Selective and dose-dependent effects of 6-AZA on expression of alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a animal model for HD. Lowering of mutant HD protein and mitigation of the "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.
Topics: Alleles; Animals; Azauridine; Cells, Cultured; DNA Repeat Expansion; Disease Models, Animal; Drosophila melanogaster; Humans; Huntingtin Protein; Huntington Disease; Luminescent Measurements; Mutant Proteins; Mutation; Nuclear Proteins; Phenotype; Photoreceptor Cells, Invertebrate; Repressor Proteins; Transcriptional Elongation Factors
PubMed: 35914128
DOI: 10.1073/pnas.2204779119 -
Journal of the American Society of... Feb 2023About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases....
BACKGROUND
About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant.
METHODS
To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models.
RESULTS
We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT.
CONCLUSIONS
Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.
Topics: Humans; Mice; Animals; Dogs; Urogenital Abnormalities; Kidney; Urinary Tract; Integrins; Mutant Proteins; Rho Guanine Nucleotide Exchange Factors
PubMed: 36414417
DOI: 10.1681/ASN.2022010050 -
JCI Insight Oct 2022Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest that selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing...
Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest that selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic protospacer adjacent motif-altering (PAM-altering) SNP (PAS), we developed an allele-specific CRISPR/Cas9 strategy to permanently inactivate mutant HTT through nonsense-mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant-specific CRISPR/Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR/Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR/Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR/Cas9 strategy inactivates mutant HTT through NMD. In addition, GUIDE-Seq analysis and subsequent validation experiments support high levels of on-target gene specificity. Our data demonstrate a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR/Cas9 and supporting its therapeutic potential in HD.
Topics: Alleles; CRISPR-Cas Systems; Gain of Function Mutation; Humans; Huntingtin Protein; Huntington Disease; Mutant Proteins; RNA, Messenger
PubMed: 36040815
DOI: 10.1172/jci.insight.141042 -
Biochemical and Biophysical Research... Dec 2022Mutations in IDH1 (isocitrate dehydrogenases) such as R132H/Q/C, are frequently found in intrahepatic cholangiocarcinoma (IHCC). Mutant IDH1 proteins obtain an abnormal...
Mutations in IDH1 (isocitrate dehydrogenases) such as R132H/Q/C, are frequently found in intrahepatic cholangiocarcinoma (IHCC). Mutant IDH1 proteins obtain an abnormal activity converting α-ketoglutarate (αKG) to 2-hydroxyglutarate (2-HG), inhibiting the activity of multiple αKG-dependent dioxygenases, leading to metabolism disorder. Here, we depict a molecular network leading by mutant IDH1, that regulates hepatic lipid embolism using mouse model (KI) with IDH1 R132Q specifically knocked in liver. KI mice appear small and have notably reduced hepatic TG and FFA levels. Technically, mutant IDH1-mediated 2-HG can stabilize PTEN mRNA level probably depending on miR-32, activate Akt-SEBP1c signaling, leading to lipogenesis defect. Our study identifies a new role of oncometabolite 2-HG in inhibiting hepatic lipid metabolism.
Topics: Mice; Animals; Lipogenesis; Liver; Mutant Proteins; Bile Duct Neoplasms; Bile Ducts, Intrahepatic
PubMed: 36410274
DOI: 10.1016/j.bbrc.2022.11.041 -
Applied and Environmental Microbiology Jan 2022Periplasmic binding proteins have been previously proclaimed as a general scaffold to design sensor proteins with new recognition specificities for nonnatural compounds....
Periplasmic binding proteins have been previously proclaimed as a general scaffold to design sensor proteins with new recognition specificities for nonnatural compounds. Such proteins can be integrated in bacterial bioreporter chassis with hybrid chemoreceptors to produce a concentration-dependent signal after ligand binding to the sensor cell. However, computationally designed new ligand-binding properties ignore the more general properties of periplasmic binding proteins, such as their periplasmic translocation, dynamic transition of open and closed forms, and interactions with membrane receptors. In order to better understand the roles of such general properties in periplasmic signaling behavior, we studied the subcellular localization of ribose-binding protein (RbsB) in Escherichia coli in comparison to a recently evolved set of mutants designed to bind 1,3-cyclohexanediol. As proxies for localization, we calibrated and deployed C-terminal end mCherry fluorescent protein fusions. Whereas RbsB-mCherry coherently localized to the periplasmic space and accumulated in (periplasmic) polar regions depending on chemoreceptor availability, mutant RbsB-mCherry expression resulted in high fluorescence cell-to-cell variability. This resulted in higher proportions of cells devoid of clear polar foci and of cells with multiple fluorescent foci elsewhere, suggesting poorer translocation, periplasmic autoaggregation, and mislocalization. Analysis of RbsB mutants and mutant libraries at different stages of directed evolution suggested overall improvement to more RbsB-wild-type-like characteristics, which was corroborated by structure predictions. Our results show that defects in periplasmic localization of mutant RbsB proteins partly explain their poor sensing performance. Future efforts should be directed to predicting or selecting secondary mutations outside computationally designed binding pockets, taking folding, translocation, and receptor interactions into account. Biosensor engineering relies on transcription factors or signaling proteins to provide the actual sensory functions for the target chemicals. Since for many compounds there are no natural sensory proteins, there is a general interest in methods that could unlock routes to obtaining new ligand-binding properties. Bacterial periplasmic binding proteins (PBPs) form an interesting family of proteins to explore for this purpose, because there is a large natural variety suggesting evolutionary trajectories to bind new ligands. PBPs are conserved and amenable to accurate computational binding pocket predictions. However, studying ribose-binding protein in Escherichia coli, we discovered that designed variants have defects in their proper localization in the cell, which can impair appropriate sensor signaling. This indicates that functional sensing capacity of PBPs cannot be obtained solely through computational design of the ligand-binding pocket but must take other properties of the protein into account, which are currently very difficult to predict.
Topics: Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Ligands; Mutant Proteins; Periplasmic Binding Proteins; Ribose
PubMed: 34757821
DOI: 10.1128/AEM.02117-21 -
Nature Nanotechnology Apr 2024In some cancers mutant p53 promotes the occurrence, development, metastasis and drug resistance of tumours, with targeted protein degradation seen as an effective...
In some cancers mutant p53 promotes the occurrence, development, metastasis and drug resistance of tumours, with targeted protein degradation seen as an effective therapeutic strategy. However, a lack of specific autophagy receptors limits this. Here, we propose the synthesis of biomimetic nanoreceptors (NRs) that mimic selective autophagy receptors. The NRs have both a component for targeting the desired protein, mutant-p53-binding peptide, and a component for enhancing degradation, cationic lipid. The peptide can bind to mutant p53 while the cationic lipid simultaneously targets autophagosomes and elevates the levels of autophagosome formation, increasing mutant p53 degradation. The NRs are demonstrated in vitro and in a patient-derived xenograft ovarian cancer model in vivo. The work highlights a possible direction for treating diseases by protein degradation.
Topics: Humans; Tumor Suppressor Protein p53; Proteolysis; Mutant Proteins; Cell Line, Tumor; Autophagy; Peptides; Lipids
PubMed: 38216684
DOI: 10.1038/s41565-023-01562-5 -
Journal of Bioscience and Bioengineering Oct 2019In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic...
In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic archaeon Sulfolobus tokodaii. The results revealed that evolvability, which is the appearance frequency of variants with higher activity than the parent protein, correlates with parental stability. This suggests that protein evolution that does not take stability into account does not make sense. Here, we used those data to further evaluate the relationship between activity and stability in random mutations, revealing that the maximum increase in activity due to mutation conflicts with parental stability. That is, many activated variants are produced when parental stability is high, whereas lower stability offers a few excellent variants with much higher activity. Moreover, we used the random mutant library to compute a novel criterion, robustizability (stabilizability), which is the appearance frequency of variants with a higher stability than the parent protein. Robustizability correlates positively with parental activity and negatively with parental stability. The results indicated that the principle of activity-stability trade-off dominates, in even random mutations. We propose its application in protein engineering via directed evolution by stability selection.
Topics: Archaeal Proteins; Enzyme Activation; Esterases; Gene Library; Mutant Proteins; Mutation; Sulfolobus
PubMed: 30987876
DOI: 10.1016/j.jbiosc.2019.03.017