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Biochemistry Jan 2021Rhodopsin is the light receptor required for the function and health of photoreceptor cells. Mutations in rhodopsin can cause misfolding and aggregation of the receptor,...
Rhodopsin is the light receptor required for the function and health of photoreceptor cells. Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinal degeneration. Bovine rhodopsin is often used as a model to understand the effect of pathogenic mutations in rhodopsin due to the abundance of structural information on the bovine form of the receptor. It is unclear whether or not the bovine rhodopsin template is adequate in predicting the effect of these mutations occurring in human retinal disease or in predicting the efficacy of therapeutic strategies. To better understand the extent to which bovine rhodopsin can serve as a model, human and bovine P23H rhodopsin mutants expressed heterologously in cells were examined. The aggregation properties and cellular localization of the mutant receptors were determined by Förster resonance energy transfer and confocal microscopy. The potential therapeutic effects of the pharmacological compounds 9- retinal and metformin were also examined. Human and bovine P23H rhodopsin mutants exhibited different aggregation properties and responses to the pharmacological compounds tested. These observations would lead to different predictions on the severity of the phenotype and divergent predictions on the benefit of the therapeutic compounds tested. The bovine rhodopsin template does not appear to adequately model the effects of the P23H mutation in the human form of the receptor.
Topics: Animals; Cattle; Diterpenes; Humans; Metformin; Mutant Proteins; Mutation; Protein Aggregates; Retinaldehyde; Rhodopsin
PubMed: 33356167
DOI: 10.1021/acs.biochem.0c00733 -
Accounts of Chemical Research Aug 2008[Figurre: see text]. Protein aggregation can be defined as the sacrifice of stabilizing intrachain contacts of the functional state that are replaced with interchain... (Comparative Study)
Comparative Study Review
[Figurre: see text]. Protein aggregation can be defined as the sacrifice of stabilizing intrachain contacts of the functional state that are replaced with interchain contacts to form non-functional states. The resulting aggregate morphologies range from amorphous structures without long-range order typical of nondisease proteins involved in inclusion bodies to highly structured fibril assemblies typical of amyloid disease proteins. In this Account, we describe the development and application of computational models for the investigation of nondisease and disease protein aggregation as illustrated for the proteins L and G and the Alzheimer's Abeta systems. In each case, we validate the models against relevant experimental observables and then expand on the experimental window to better elucidate the link between molecular properties and aggregation outcomes. Our studies show that each class of protein exhibits distinct aggregation mechanisms that are dependent on protein sequence, protein concentration, and solution conditions. Nondisease proteins can have native structural elements in the denatured state ensemble or rapidly form early folding intermediates, which offers avenues of protection against aggregation even at relatively high concentrations. The possibility that early folding intermediates may be evolutionarily selected for their protective role against unwanted aggregation could be a useful strategy for reengineering sequences to slow aggregation and increase folding yield in industrial protein production. The observed oligomeric aggregates that we see for nondisease proteins L and G may represent the nuclei for larger aggregates, not just for large amorphous inclusion bodies, but potentially as the seeds of ordered fibrillar aggregates, since most nondisease proteins can form amyloid fibrils under conditions that destabilize the native state. By contrast, amyloidogenic protein sequences such as Abeta 1-40,42 and the familial Alzheimer's disease (FAD) mutants favor aggregation into ordered fibrils once the free-energy barrier for forming a critical nucleus is crossed. However, the structural characteristics and oligomer size of the soluble nucleation species have yet to be determined experimentally for any disease peptide sequence, and the molecular mechanism of polymerization that eventually delineates a mature fibril is unknown. This is in part due to the limited experimental access to very low peptide concentrations that are required to characterize these early aggregation events, providing an opportunity for theoretical studies to bridge the gap between the monomer and fibril end points and to develop testable hypotheses. Our model shows that Abeta 1-40 requires as few as 6-10 monomer chains (depending on sequence) to begin manifesting the cross-beta order that is a signature of formation of amyloid filaments or fibrils assessed in dye-binding kinetic assays. The richness of the oligomeric structures and viable filament and fibril polymorphs that we observe may offer structural clues to disease virulence variations that are seen for the WT and hereditary mutants.
Topics: Alzheimer Disease; Animals; Humans; Models, Molecular; Mutant Proteins; Protein Binding; Protein Conformation; Protein Folding
PubMed: 18646868
DOI: 10.1021/ar800062k -
Biochemistry Sep 2009Conformational change in the prion protein (PrP) is thought to be responsible for a group of rare but fatal neurodegenerative diseases of humans and other animals,...
Conformational change in the prion protein (PrP) is thought to be responsible for a group of rare but fatal neurodegenerative diseases of humans and other animals, including Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. However, little is known about the mechanism by which normal cellular PrPs initiate and propagate the conformational change. Here, we studied backbone dynamics of the inherited pathogenic mutants (P101L and H186R), protective mutants (Q167R and Q218K), and wild-type mouse PrP(89-230) at pH 5.5 and 3.5. Mutations result in minor chemical shift changes around the mutation sites except that H186R induces large chemical shift changes at distal regions. At lower pH values, the C-terminal half of the second helix is significantly disordered for the wild-type and all mutant proteins, while other parts of the protein are essentially unaffected. This destabilization is accompanied by protonation of the partially exposed histidine H186 in the second helix of the wild-type protein. This region in the mutant protein H186R is disordered even at pH 5.5. The wild-type and mutant proteins have similar microsecond conformational exchange near the two beta-strands and have similar nanosecond internal motions in several regions including the C-terminal half of the second helix, but only wild type and P101L have extensive nanosecond internal motions throughout the helices. These motions mostly disappear at lower pH. Our findings raise the possibility that the pathogenic or dominant negative mutations exert their effects on some non-native intermediate form such as PrP* after conversion of cellular PrP (PrP(C)) into the pathogenic isoform PrP(Sc) has been initiated; additionally, formation of PrP(Sc) might begin within the C-terminal folded region rather than in the disordered N-terminal region.
Topics: Amino Acid Sequence; Animals; Humans; Hydrogen-Ion Concentration; Mice; Models, Molecular; Molecular Sequence Data; Mutant Proteins; Mutation; Neurodegenerative Diseases; Prions; Protein Conformation; Protein Folding; Protons
PubMed: 19618915
DOI: 10.1021/bi900923b -
Cells Nov 2021The wild-type protein p53 plays a key role in preventing the formation of neoplasms by controlling cell growth. However, in more than a half of all cancers, the gene... (Review)
Review
The wild-type protein p53 plays a key role in preventing the formation of neoplasms by controlling cell growth. However, in more than a half of all cancers, the gene has missense mutations that appear during tumorigenesis. In most cases, the mutated gene encodes a full-length protein with the substitution of a single amino acid, resulting in structural and functional changes and acquiring an oncogenic role. This dual role of the wild-type protein and the mutated isoforms is also evident in the regulation of the redox state of the cell, with antioxidant and prooxidant functions, respectively. In this review, we introduce a new concept of the p53 protein by discussing its sensitivity to the cellular redox state. In particular, we focus on the discussion of structural and functional changes following post-translational modifications of redox-sensitive cysteine residues, which are also responsible for interacting with zinc ions for proper structural folding. We will also discuss therapeutic opportunities using small molecules targeting cysteines capable of modifying the structure and function of the p53 mutant isoforms in view of possible anticancer therapies for patients possessing the mutation in the gene.
Topics: Animals; Cysteine; Humans; Mutant Proteins; Oxidation-Reduction; Protein Isoforms; Protein Processing, Post-Translational; Structure-Activity Relationship; Tumor Suppressor Protein p53
PubMed: 34831372
DOI: 10.3390/cells10113149 -
PloS One 2020Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder,...
Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.
Topics: Amino Acid Substitution; Autoimmune Diseases; Glycosylation; Humans; Mutant Proteins; Mutation, Missense; Osteochondrodysplasias; Proteolysis; Tartrate-Resistant Acid Phosphatase
PubMed: 32214327
DOI: 10.1371/journal.pone.0230052 -
Biomolecules Nov 2020The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research,... (Review)
Review
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, mutants lacking (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.
Topics: Bacterial Proteins; DNA-Directed RNA Polymerases; Mutant Proteins; Protein Subunits; Structure-Activity Relationship; Transcription Factors
PubMed: 33238579
DOI: 10.3390/biom10111588 -
BMC Microbiology Oct 2013Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV...
BACKGROUND
Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication.
RESULTS
To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase.
CONCLUSIONS
C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.
Topics: Animals; Bacterial Proteins; Chlorocebus aethiops; Computational Biology; Coxiella burnetii; Culture Media; Mass Spectrometry; Metabolic Networks and Pathways; Mutant Proteins; Protein Sorting Signals; Protein Transport; Sequence Deletion; Vero Cells
PubMed: 24093460
DOI: 10.1186/1471-2180-13-222 -
Journal of Thrombosis and Haemostasis :... Nov 2011Substitutive therapy has significantly ameliorated the quality of life of patients with coagulation factor deficiencies. However, there are some limitations that support... (Review)
Review
Substitutive therapy has significantly ameliorated the quality of life of patients with coagulation factor deficiencies. However, there are some limitations that support research towards alternative therapeutic approaches. Here we focus on the rescue of coagulation factor biosynthesis by targeting the RNA processing and translation, which would permit restoration of the altered gene expression while maintaining the gene regulation in the physiological tissues. The essential prerequisite of the three reported RNA-based correction approaches (i-iii), which rely on mutation types and are applicable even to large size mRNAs, is the presence in cells of the precursor (pre-mRNA) or mature mRNA forms. (i) In the F7 gene, modification of the small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, re-directs correct usage of a mutated exon-intron junction, triggering synthesis of correct mRNA and secretion of functional factor (F)VII. (ii) Spliceosome-mediated RNA trans-splicing (SMaRT) between mutated and engineered pre-mRNAs produces normal FVIII mRNA and secretion of functional protein. (iii) Aminoglycoside drugs induce ribosome readthrough and suppress premature translation termination caused by nonsense mutations in FVII, VIII and IX. The rescued expression levels ranged from very low (aminoglycosides) to moderate (U1 snRNA and SMaRT), which could result in amelioration of the disease phenotypes. These findings prompt further studies aimed at demonstrating the clinical translatability of RNA-based strategies, which might open new avenues in the treatment of coagulation factor deficiencies.
Topics: Blood Coagulation Factors; Coagulation Protein Disorders; Genetic Therapy; Humans; Mutant Proteins; RNA; RNA Splicing
PubMed: 21854538
DOI: 10.1111/j.1538-7836.2011.04481.x -
Blood Jan 2011After the discovery of NPM1-mutated acute myeloid leukemia (AML) in 2005 and its subsequent inclusion as a provisional entity in the 2008 World Health Organization... (Review)
Review
After the discovery of NPM1-mutated acute myeloid leukemia (AML) in 2005 and its subsequent inclusion as a provisional entity in the 2008 World Health Organization classification of myeloid neoplasms, several controversial issues remained to be clarified. It was unclear whether the NPM1 mutation was a primary genetic lesion and whether additional chromosomal aberrations and multilineage dysplasia had any impact on the biologic and prognostic features of NPM1-mutated AML. Moreover, it was uncertain how to classify AML patients who were double-mutated for NPM1 and CEBPA. Recent studies have shown that: (1) the NPM1 mutant perturbs hemopoiesis in experimental models; (2) leukemic stem cells from NPM1-mutated AML patients carry the mutation; and (3) the NPM1 mutation is usually mutually exclusive of biallelic CEPBA mutations. Moreover, the biologic and clinical features of NPM1-mutated AML do not seem to be significantly influenced by concomitant chromosomal aberrations or multilineage dysplasia. Altogether, these pieces of evidence point to NPM1-mutated AML as a founder genetic event that defines a distinct leukemia entity accounting for approximately one-third of all AML.
Topics: Animals; Founder Effect; Humans; Leukemia, Myeloid, Acute; Models, Biological; Mutant Proteins; Mutation; Nuclear Proteins; Nucleophosmin; Prognosis
PubMed: 21030560
DOI: 10.1182/blood-2010-08-299990 -
Journal of Molecular Biology Aug 2017Influenza virus evolves rapidly to constantly escape from natural immunity. Most humoral immune responses to influenza virus target the hemagglutinin (HA) glycoprotein,... (Review)
Review
Influenza virus evolves rapidly to constantly escape from natural immunity. Most humoral immune responses to influenza virus target the hemagglutinin (HA) glycoprotein, which is the major antigen on the surface of the virus. The HA is composed of a globular head domain for receptor binding and a stem domain for membrane fusion. The major antigenic sites of HA are located in the globular head subdomain, which is highly tolerant of amino acid substitutions and continual addition of glycosylation sites. Nonetheless, the evolution of the receptor-binding site and the stem region on HA is severely constrained by their functional roles in engaging the host receptor and in mediating membrane fusion, respectively. Here, we review how broadly neutralizing antibodies (bnAbs) exploit these evolutionary constraints to protect against diverse influenza strains. We also discuss the emerging role of other epitopes that are conserved only in subsets of viruses. This rapidly increasing knowledge of the evolutionary biology, immunology, structural biology, and virology of influenza virus is invaluable for development and design of more universal influenza vaccines and novel therapeutics.
Topics: Antibodies, Neutralizing; Antibodies, Viral; Evolution, Molecular; Hemagglutinin Glycoproteins, Influenza Virus; Immune Evasion; Mutant Proteins; Mutation; Orthomyxoviridae
PubMed: 28648617
DOI: 10.1016/j.jmb.2017.06.015