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Cells May 2020Brain-derived neurotrophic factor (BDNF), a major focus for regenerative therapeutics, has been lauded for its pro-survival characteristics and involvement in both... (Review)
Review
Brain-derived neurotrophic factor (BDNF), a major focus for regenerative therapeutics, has been lauded for its pro-survival characteristics and involvement in both development and recovery of function within the central nervous system (CNS). However, studies of tyrosine receptor kinase B (TrkB), a major receptor for BDNF, indicate that certain effects of the TrkB receptor in response to disease or injury may be maladaptive. More specifically, imbalance among TrkB receptor isoforms appears to contribute to aberrant signaling and hyperpathic pain. A truncated isoform of the receptor, TrkB.T1, lacks the intracellular kinase domain of the full length receptor and is up-regulated in multiple CNS injury models. Such up-regulation is associated with hyperpathic pain, and TrkB.T1 inhibition reduces neuropathic pain in various experimental paradigms. Deletion of TrkB.T1 also limits astrocyte changes in vitro, including proliferation, migration, and activation. Mechanistically, TrkB.T1 is believed to act through release of intracellular calcium in astrocytes, as well as through interactions with neurotrophins, leading to cell cycle activation. Together, these studies support a potential role for astrocytic TrkB.T1 in hyperpathic pain and suggest that targeted strategies directed at this receptor may have therapeutic potential.
Topics: Animals; Brain-Derived Neurotrophic Factor; Embryonic Development; Humans; Mutant Proteins; Neuralgia; Receptor, trkB; Signal Transduction
PubMed: 32403409
DOI: 10.3390/cells9051194 -
Redox Biology Oct 2023The lens proteome undergoes dramatic composition changes during development and maturation. A defective developmental process leads to congenital cataracts that account...
The lens proteome undergoes dramatic composition changes during development and maturation. A defective developmental process leads to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early-onset forms of lens opacity, with the remainder being of unknown etiology. To gain a better understanding of cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W-Ub) that recapitulates most of the early pathological changes seen in human congenital cataracts. We performed mass spectrometry-based tandem-mass-tag quantitative proteomics in E15, P1, and P30 control or K6W-Ub lenses. Our analysis identified targets that are required for early normal differentiation steps and altered in cataractous lenses, particularly metabolic pathways involving glutathione and amino acids. Computational molecular phenotyping revealed that glutathione and taurine were spatially altered in the K6W-Ub cataractous lens. High-performance liquid chromatography revealed that both taurine and the ratio of reduced glutathione to oxidized glutathione, two indicators of redox status, were differentially compromised in lens biology. In sum, our research documents that dynamic proteome changes in a mouse model of congenital cataracts impact redox biology in lens. Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age-related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.
Topics: Humans; Animals; Mice; Proteome; Glutathione; Disease Models, Animal; Mice, Transgenic; Mutant Proteins; Oxidation-Reduction; Taurine; Cataract
PubMed: 37677999
DOI: 10.1016/j.redox.2023.102869 -
Cells Sep 2022(1) Background: Activity-dependent neuroprotective protein (ADNP) is essential for neuronal structure and function. Multiple de novo pathological mutations in ADNP cause...
(1) Background: Activity-dependent neuroprotective protein (ADNP) is essential for neuronal structure and function. Multiple de novo pathological mutations in ADNP cause the autistic ADNP syndrome, and they have been further suggested to affect Alzheimer's disease progression in a somatic form. Here, we asked if different ADNP mutations produce specific neuronal-like phenotypes toward better understanding and personalized medicine. (2) Methods: We employed CRISPR/Cas9 genome editing in N1E-115 neuroblastoma cells to form neuron-like cell lines expressing ADNP mutant proteins conjugated to GFP. These new cell lines were characterized by quantitative morphology, immunocytochemistry and live cell imaging. (3) Results: Our novel cell lines, constitutively expressing GFP-ADNP p.Pro403 (p.Ser404* human orthologue) and GFP-ADNP p.Tyr718* (p.Tyr719* human orthologue), revealed new and distinct phenotypes. Increased neurite numbers (day 1, in culture) and increased neurite lengths upon differentiation (day 7, in culture) were linked with p.Pro403*. In contrast, p.Tyr718* decreased cell numbers (day 1). These discrete phenotypes were associated with an increased expression of both mutant proteins in the cytoplasm. Reduced nuclear/cytoplasmic boundaries were observed in the p.Tyr718* ADNP-mutant line, with this malformation being corrected by the ADNP-derived fragment drug candidate NAP. (4) Conclusions: Distinct impairments characterize different ADNP mutants and reveal aberrant cytoplasmic-nuclear crosstalk.
Topics: Autistic Disorder; Cytoplasm; Homeodomain Proteins; Humans; Mutant Proteins; Nerve Tissue Proteins
PubMed: 36230962
DOI: 10.3390/cells11192994 -
Genes To Cells : Devoted To Molecular &... Jun 202114-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between...
14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.
Topics: 14-3-3 Proteins; Amino Acid Sequence; Binding Sites; Centrioles; Centrosome; HCT116 Cells; HEK293 Cells; Humans; Models, Biological; Mutant Proteins; Nuclear Proteins; Nucleophosmin; Phenotype; Phosphopeptides; Phosphorylation; Protein Multimerization; rho-Associated Kinases
PubMed: 33813791
DOI: 10.1111/gtc.12848 -
Bioconjugate Chemistry Sep 2023Protein arylation has attracted much attention for developing new classes of bioconjugates with improved properties. Here, we have evaluated 2-sulfonylpyrimidines as...
Protein arylation has attracted much attention for developing new classes of bioconjugates with improved properties. Here, we have evaluated 2-sulfonylpyrimidines as covalent warheads for the mild, chemoselective, and metal free cysteine -arylation. 2-Sulfonylpyrimidines react rapidly with cysteine, resulting in stable -heteroarylated adducts at neutral pH. Fine tuning the heterocyclic core and exocyclic leaving group allowed predictable SAr reactivity , covering >9 orders of magnitude. Finally, we achieved fast chemo- and regiospecific arylation of a mutant p53 protein and confirmed arylation sites by protein X-ray crystallography. Hence, we report the first example of a protein site specifically -arylated with iodo-aromatic motifs. Overall, this study provides the most comprehensive structure-reactivity relationship to date on heteroaryl sulfones and highlights 2-sulfonylpyrimidine as a synthetically tractable and protein compatible covalent motif for targeting reactive cysteines, expanding the arsenal of tunable warheads for modern covalent ligand discovery.
Topics: Cysteine; Mutant Proteins; Crystallography, X-Ray; Sulfones
PubMed: 37657082
DOI: 10.1021/acs.bioconjchem.3c00322 -
Nucleic Acids Research Jan 2021ProThermDB is an updated version of the thermodynamic database for proteins and mutants (ProTherm), which has ∼31 500 data on protein stability, an increase of 84%...
ProThermDB is an updated version of the thermodynamic database for proteins and mutants (ProTherm), which has ∼31 500 data on protein stability, an increase of 84% from the previous version. It contains several thermodynamic parameters such as melting temperature, free energy obtained with thermal and denaturant denaturation, enthalpy change and heat capacity change along with experimental methods and conditions, sequence, structure and literature information. Besides, the current version of the database includes about 120 000 thermodynamic data obtained for different organisms and cell lines, which are determined by recent high throughput proteomics techniques using whole-cell approaches. In addition, we provided a graphical interface for visualization of mutations at sequence and structure levels. ProThermDB is cross-linked with other relevant databases, PDB, UniProt, PubMed etc. It is freely available at https://web.iitm.ac.in/bioinfo2/prothermdb/index.html without any login requirements. It is implemented in Python, HTML and JavaScript, and supports the latest versions of major browsers, such as Firefox, Chrome and Safari.
Topics: Databases, Protein; Information Storage and Retrieval; Mutant Proteins; Proteins; Statistics as Topic; Thermodynamics
PubMed: 33196841
DOI: 10.1093/nar/gkaa1035 -
Advanced Science (Weinheim,... Nov 2023Accumulation of misfolded proteins leads to many neurodegenerative diseases that can be treated by lowering or removing mutant proteins. Huntington's disease (HD) is...
Accumulation of misfolded proteins leads to many neurodegenerative diseases that can be treated by lowering or removing mutant proteins. Huntington's disease (HD) is characterized by the intracellular accumulation of mutant huntingtin (mHTT) that can be soluble and aggregated in the central nervous system and causes neuronal damage and death. Here, an intracellular antibody (intrabody) fragment is generated that can specifically bind mHTT and link to the lysosome for degradation. It is found that delivery of this peptide by either brain injection or intravenous administration can efficiently clear the soluble and aggregated mHTT by activating the lysosomal degradation pathway, resulting in amelioration of gliosis and dyskinesia in HD knock-in (KI-140Q) mice. These findings suggest that the small intrabody peptide linked to lysosomes can effectively lower mutant proteins and provide a new approach for treating neurodegenerative diseases that are caused by the accumulation of mutant proteins.
Topics: Animals; Mice; Huntingtin Protein; Huntington Disease; Lysosomes; Mutant Proteins; Nerve Tissue Proteins; Neurodegenerative Diseases; Peptides
PubMed: 37688357
DOI: 10.1002/advs.202301120 -
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