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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 -
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 -
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 Communications Mar 2020Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to...
Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to these response differences, here we analyze in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis indicates that the p53 family protein, p73, affects Th1 differentiation. In cells differentiated under Th1 conditions in vitro, p73 negatively regulates IFNγ production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes such as Ifng and Il12rb2. Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice have increased IFNγ production and less disease severity, whereas in an adoptive transfer model of inflammatory bowel disease, transfer of p73-deficient naïve CD4 T cells increases Th1 responses and augments disease severity. Our results thus identify p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease.
Topics: Alleles; Animals; Base Sequence; Binding Sites; Cell Differentiation; Colitis; DNA; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Deletion; Gene Expression Regulation; Interferon-gamma; Mice; Mutant Proteins; Protein Binding; Protein Domains; Severity of Illness Index; Th1 Cells; Tumor Protein p73; Tumor Suppressor Protein p53
PubMed: 32193462
DOI: 10.1038/s41467-020-15172-5 -
Proceedings of the National Academy of... Dec 2019Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the...
Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies.
Topics: Aging; Amyotrophic Lateral Sclerosis; Gene Expression Regulation; Gene Knockdown Techniques; HEK293 Cells; Humans; Inclusion Bodies; Molecular Weight; Mutant Proteins; Mutation; Saccharomyces cerevisiae; Superoxide Dismutase-1
PubMed: 31796595
DOI: 10.1073/pnas.1902483116 -
Neurobiology of Disease May 2020Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common...
Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-β1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-β1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-β1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis.
Topics: Alternative Splicing; Amyotrophic Lateral Sclerosis; Animals; C9orf72 Protein; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Humans; Mice; Motor Neurons; Mutant Proteins; Mutation; Protein Transport; RNA-Binding Protein FUS; Ribonucleoproteins, Small Nuclear
PubMed: 32027933
DOI: 10.1016/j.nbd.2020.104792 -
Proceedings of the National Academy of... Aug 2019Mutant huntingtin (mHTT), the causative protein in Huntington's disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex,...
Mutant huntingtin (mHTT), the causative protein in Huntington's disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space. We investigated differences in mitochondrial proteome between wtHTT and mHTT cells and found numerous proteomic disparities between mHTT and wtHTT mitochondria. We validated these data by quantitative immunoblotting in striatal cell lines and human HD brain tissue. The level of soluble matrix mitochondrial proteins imported through the TIM23 complex is lower in mHTT-expressing cell lines and brain tissues of HD patients compared with controls. In mHTT-expressing cell lines, membrane-bound TIM23-imported proteins have lower intramitochondrial levels, whereas inner membrane multispan proteins that are imported via the TIM22 pathway and proteins integrated into the outer membrane generally remain unchanged. In summary, we show that, in mitochondria, huntingtin is located in the intermembrane space, that mHTT binds with high-affinity to TIM23, and that mitochondria from mHTT-expressing cells and brain tissues of HD patients have reduced levels of nuclearly encoded proteins imported through TIM23. These data demonstrate the mechanism and biological significance of mHTT-mediated inhibition of mitochondrial protein import, a mechanism likely broadly relevant to other neurodegenerative diseases.
Topics: Cell Line; Cell Nucleus; Cerebral Cortex; Corpus Striatum; Humans; Huntingtin Protein; Huntington Disease; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Mutant Proteins; Protein Binding; Proteome; Proteostasis
PubMed: 31346086
DOI: 10.1073/pnas.1904101116