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Characterization of Novel Missense Variants of SERPINA1 Gene Causing Alpha-1 Antitrypsin Deficiency.American Journal of Respiratory Cell... Jun 2018The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe...
The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.
Topics: Adult; Aged; Female; Gene Frequency; HEK293 Cells; Humans; Male; Middle Aged; Mutant Proteins; Mutation, Missense; Protein Stability; Proteolysis; alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency
PubMed: 29232161
DOI: 10.1165/rcmb.2017-0179OC -
The Biochemical Journal Jun 20214-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism....
4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.
Topics: 4-Hydroxyphenylpyruvate Dioxygenase; Catalysis; Humans; Kinetics; Ligands; Models, Molecular; Mutant Proteins; Mutation; Protein Conformation; Substrate Specificity
PubMed: 34047349
DOI: 10.1042/BCJ20210005 -
International Journal of Molecular... Jan 2021Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to...
BACKGROUND
Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK-RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model.
RESULTS
As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.
Topics: Animals; Cells, Cultured; Female; Mice; Mice, Inbred C57BL; Mutant Proteins; Mutation; Osteoclasts; Osteogenesis; RANK Ligand; Signal Transduction
PubMed: 33406741
DOI: 10.3390/ijms22010434 -
Critical Reviews in Biochemistry and... 2015Most proteins have multiple functions. Obviously, conventional methods of manipulating the level of the protein of interest in the cell, such as over-expression,... (Review)
Review
Most proteins have multiple functions. Obviously, conventional methods of manipulating the level of the protein of interest in the cell, such as over-expression, knockout or knockdown, affect all of its functions simultaneously. The key advantage of these methods is that over-expression, knockout or knockdown does not require any knowledge of the molecular mechanisms of the function(s) of the protein of interest. The disadvantage is that these approaches are inadequate to elucidate the role of an individual function of the protein in a particular cellular process. An alternative is the use of re-engineered proteins, in which a single function is eliminated or enhanced. The use of mono-functional elements of a multi-functional protein can also yield cleaner answers. This approach requires detailed knowledge of the structural basis of each function of the protein in question. Thus, a lot of preliminary structure-function work is necessary to make it possible. However, when this information is available, replacing the protein of interest with a mutant in which individual functions are modified can shed light on the biological role of those particular functions. Here, we illustrate this point using the example of protein kinases, most of which have additional non-enzymatic functions, as well as arrestins, known multi-functional signaling regulators in the cell.
Topics: Animals; Arrestins; Enzyme Activation; G-Protein-Coupled Receptor Kinases; Gene Knockdown Techniques; Gene Knockout Techniques; Humans; Ligands; Models, Molecular; Mutant Proteins; Protein Conformation; Recombinant Proteins; Signal Transduction
PubMed: 26453028
DOI: 10.3109/10409238.2015.1067185 -
International Journal of Biological... May 2023Stability is critical for the proper functioning of all proteins. Optimization of protein thermostability is a key step in the development of industrial enzymes and...
Stability is critical for the proper functioning of all proteins. Optimization of protein thermostability is a key step in the development of industrial enzymes and biologics. Herein, we demonstrate that multidomain proteins can be stabilized significantly using domain-based engineering followed by the recombination of the optimized domains. Domain-level analysis of designed protein variants with similar structures but different thermal profiles showed that the independent enhancement of the thermostability of a constituent domain improves the overall stability of the whole multidomain protein. The crystal structure and AlphaFold-predicted model of the designed proteins via domain-recombination provided a molecular explanation for domain-based stepwise stabilization. Our study suggests that domain-based modular engineering can minimize the sequence space for calculations in computational design and experimental errors, thereby offering useful guidance for multidomain protein engineering.
Topics: Proteins; Mutant Proteins; Enzyme Stability
PubMed: 36958447
DOI: 10.1016/j.ijbiomac.2023.124141 -
Nature Sep 2015The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes....
The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction.
Topics: Charcot-Marie-Tooth Disease; Crystallography, X-Ray; Dynamins; Humans; Markov Chains; Models, Molecular; Molecular Dynamics Simulation; Mutant Proteins; Mutation; Myopathies, Structural, Congenital; Nucleotides; Protein Multimerization; Structure-Activity Relationship
PubMed: 26302298
DOI: 10.1038/nature14880 -
The Journal of Physical Chemistry. B Jan 2024Determining changes in the protein's thermal stability following mutations is critical in protein engineering and understanding pathogenic missense mutations. Despite...
Determining changes in the protein's thermal stability following mutations is critical in protein engineering and understanding pathogenic missense mutations. Despite the development of various computational methods to predict the effects of single-point mutations, their accuracy remains limited. In this study, we propose a new computational method, OmeDDG, that more accurately predicts mutation-induced Gibbs free energy changes in protein folding (ΔΔ). OmeDDG takes the sequences of wild-type and mutant proteins as input, utilizes OmegaFold to obtain the 3D structure, employs a convolutional neural network to extract structural features, and combines them with protein mutation features and pretraining features to predict the stability of single-point mutations in proteins. We performed a comprehensive comparison between OmeDDG and other available prediction methods on four blind test datasets, confirming that OmeDDG can effectively enhance protein mutation prediction performance. Notably, on the antisymmetric dataset Ssym, OmeDDG achieves the best performance, demonstrating favorable antisymmetry with PCC = 0.79 and RMSE = 0.96 for forward mutations and PCC = 0.77 and RMSE = 0.97 for reverse mutant types.
Topics: Mutation; Point Mutation; Proteins; Mutant Proteins; Protein Folding; Protein Stability
PubMed: 38130113
DOI: 10.1021/acs.jpcb.3c05601 -
Journal of Virology Aug 2017Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown,...
Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity. Despite the availability of effective vaccines, both measles virus (MeV) and canine distemper virus (CDV) still lead to significant human and animal mortality worldwide. It is assumed that postexposure prophylaxis with specific antiviral compounds may synergize with vaccination campaigns to better control ongoing epidemics. Targeting the matrix (M) protein of MeV/CDV is attractive, because M coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, the lack of basic molecular knowledge of how M orchestrates these functions precludes the rational design of antivirals. Here we combined structure-guided mutagenesis with cellular, biochemical, and functional assays to investigate a potential correlation between CDV M self-assembly and virus-like particle (VLP) formation. Altogether, our findings provide evidence that stable M dimers at the cell periphery are required to productively trigger VLPs. Such stabilized M dimeric units may facilitate further assembly into robust higher-order oligomers necessary to promote plasma membrane-budding activity.
Topics: DNA Mutational Analysis; Distemper Virus, Canine; Mutant Proteins; Protein Multimerization; Viral Matrix Proteins; Virus Release
PubMed: 28592541
DOI: 10.1128/JVI.00521-17 -
Scientific Reports Oct 2018Phosphoinositide 3-kinase alpha (PI3Kα) is involved in fundamental cellular processes including cell proliferation and differentiation and is frequently mutated in...
Phosphoinositide 3-kinase alpha (PI3Kα) is involved in fundamental cellular processes including cell proliferation and differentiation and is frequently mutated in human malignancies. One of the most common mutations is E545K, which results in an amino acid substitution of opposite charge. It has been recently proposed that in this oncogenic charge-reversal mutation, the interactions between the protein catalytic and regulatory subunits are abrogated, resulting in loss of regulation and constitutive PI3Kα activity, which can lead to oncogenesis. To assess the mechanism of the PI3Kα E545K activating mutation, extensive Molecular Dynamics simulations were performed to examine conformational changes differing between the wild type (WT) and mutant proteins as they occur in microsecond simulations. In the E545K mutant PI3Kα, we observe a spontaneous detachment of the nSH2 PI3Kα domain (regulatory subunit, p85α) from the helical domain (catalytic subunit, p110α) causing significant loss of communication between the regulatory and catalytic subunits. We examine the allosteric network of the two proteins and show that a cluster of residues around the mutation is important for delivering communication signals between the catalytic and regulatory subunits. Our results demonstrate the dynamical and structural effects induced by the p110α E545K mutation in atomic level detail and indicate a possible mechanism for the loss of regulation that E545K confers on PI3Kα.
Topics: Amino Acid Substitution; Class I Phosphatidylinositol 3-Kinases; Enzyme Activation; Humans; Molecular Dynamics Simulation; Mutant Proteins; Mutation, Missense; Protein Conformation
PubMed: 30341384
DOI: 10.1038/s41598-018-27044-6 -
Methods in Molecular Biology (Clifton,... 2015Gli2 and Gli3 respond to the Hedgehog (Hh) signal in mammals by undergoing posttranslational modifications and moving to the nucleus. The study of Gli proteins has been...
Gli2 and Gli3 respond to the Hedgehog (Hh) signal in mammals by undergoing posttranslational modifications and moving to the nucleus. The study of Gli proteins has been hampered by the fact that their overexpression in cells prevents their proper regulation. To address this issue, we have developed a method of rapid generation of stable cell lines expressing near-endogenous and approximately equal levels of wild-type and mutant Gli proteins. This method is applicable to the study of effects of various mutations on Gli protein modifications and activity.
Topics: 3T3 Cells; Animals; Gene Expression; Homologous Recombination; Kruppel-Like Transcription Factors; Mice; Mutant Proteins; Mutation; Nerve Tissue Proteins; Transfection; Zinc Finger Protein Gli2; Zinc Finger Protein Gli3
PubMed: 26179044
DOI: 10.1007/978-1-4939-2772-2_11