Stability Of Mutant Proteins Research Articles

Computational Analysis of Boerhavia diffusa Plant Extracts Targeting Alpha actinin4 against Focal Segmental Glomerulosclerosis

The most common protein lost in urine (proteinuria) is albumin, which is a symptom of the kidney illness focal segmental glomerulosclerosis (FSGS). It causes damage to podocyte foot processes, resulting in effacement of foot processes, and injury to foot processes leads to the leaking of plasma proteins into the urine, resulting in nephrotic syndrome. Alpha actinin (ACTN4) is extensively produced in glomerular podocytes, has a function in non-muscle cytoskeletal activity, and is enhanced early in the course of nephrotic illness in various types. Mutations in ACTN4 induce autosomal and sporadic steroid-resistance nephritic syndrome, which causes disturbance in podocyte foot process and function. All of the FSGS-causing mutations are situated on actin actin-binding domain of the ACTN4 protein. FSGS is typically treated by decreasing dietary salt and using immune-suppressing medications such as glucocorticoids. There is a risk associated with these drugs for cancer patients. Several studies have found that up to 80% of individuals with primary FSGS are resistant to steroid therapy, even though all other therapeutic options have been exhausted. Patients with steroid-resistant FSGS have a higher incidence of end-stage renal failure. Therefore, several new treatment strategies were put forward to cure the disease form of which use of phytochemicals is one such sustainable modality. In addition to this, our study intended to predict the stability of mutant protein’s structure as compared to wild type structure through molecular dynamic simulation analysis. With this, we extend our study to predict the potency of phytochemicals of Boerhaviadiffusa against FSGS by using molecular docking analysis. From our study, we conclude that the actin-binding domain of ACTN4 protein becomes more unstable or loses its stability after the mutation at position W59R among all the studied mutation positions. The boeravinone F phytochemical of the Boerhaviadiffusa plant shows the best inhibitory effect on the mutant actin-binding domain of ACTN4 protein and it confirms a stable binding confirmation at minimum energy of -7.5 kcal/mol. Hence it may be taken into consideration for future research work.

GEN1 as a risk factor for human congenital anomalies of the kidney and urinary tract

BackgroundCongenital anomalies of the kidney and urinary tract (CAKUT) are prevalent birth defects. Although pathogenic CAKUT genes are known, they are insufficient to reveal the causes for all patients. Our previous studies indicated GEN1 as a pathogenic gene of CAKUT in mice, and this study further investigated the correlation between GEN1 and human CAKUT.MethodsIn this study, DNA from 910 individuals with CAKUT was collected; 26 GEN1 rare variants were identified, and two GEN1 (missense) variants in a non-CAKUT group were found. Mainly due to the stability results of the predicted mutant on the website, in vitro, 10 variants (eight CAKUT, two non-CAKUT) were selected to verify mutant protein stability. In addition, mainly based on the division of the mutation site located in the functional region of the GEN1 protein, 8 variants (six CAKUT, two non-CAKUT) were selected to verify enzymatic hydrolysis, and the splice variant GEN1 (c.1071 + 3(IVS10) A > G) was selected to verify shear ability. Based on the results of in vitro experiments and higher frequency, three sites with the most significant functional change were selected to build mouse models.ResultsProtein stability changed in six variants in the CAKUT group. Based on electrophoretic mobility shift assay of eight variants (six CAKUT, two non-CAKUT), the enzymatic hydrolysis and DNA-binding abilities of mutant proteins were impaired in the CAKUT group. The most serious functional damage was observed in the Gen1 variant that produced a truncated protein. A mini-gene splicing assay showed that the variant GEN1 (c.1071 + 3(IVS10) A > G) in the CAKUT group significantly affected splicing function. An abnormal exon10 was detected in the mini-gene splicing assay. Point-mutant mouse strains were constructed (Gen1: c.1068 + 3 A > G, p.R400X, and p.T105R) based on the variant frequency in the CAKUT group and functional impairment in vitro study and CAKUT phenotypes were replicated in each.ConclusionOverall, our findings indicated GEN1 as a risk factor for human CAKUT.

Targeting the Bap1 protein of Vibrio cholerae for screening potent inhibitors and predicting the mutant protein stability: in silico analyses

Background: Bap1 is reported to be a major protein in Vibrio cholerae which aids in biofilm formation. Hence, inhibition of the protein molecule can no longer support the colonization of bacteria and induction of point mutations at specific amino acid residues tend to reduce the protein stabilization. The aim of the study: To target this Bap1 structural protein for screening potent inhibitors of ligands by means of in silico analyses. Materials and methods: A total of 30 compounds divided into three groups such as synthetic antimicrobial drugs, phytochemicals and marine compounds comprising of ten ligands in each group were tested against Bap1. In addition to this, mutations were induced at GLN: 518, HIS: 520 and ASN: 679 positions to determine the stability of the mutant Bap1 protein using bioinformatic tools. Results: Of the 30 docked compounds, doxycycline, ichangin and Ageloxime D exhibited the highest binding affinities of -8.5 kcal/mol, -9.3 kcal/mol and -8.8 kcal/mol respectively from the three groups. The ADME properties show the druglikeness of the test compounds to be used for treatment procedures. Protein-ligand interactions were visualized which infer that both doxycycline and ichangin form five conventional hydrogen bonds while Ageloxime D could form three hydrogen bonds with different amino acid residues of the protein. Further, the van der Waals’ interactions are also found to be similar in number among doxycycline and ichangin whereas, it is less in case of Ageloxime D but, the π- interactions are high in this compound comparatively. The carbon-hydrogen bonding for these three compounds with the amino acid residues of the Bap1 protein have also been discussed. Conclusion: Thus, natural compounds can eventually replace the over use of synthetic drugs as antibiofilm agents. Also, inducing point mutations to this protein can potentially destabilize its structure.

The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA.

Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel β-strands, offset by an increase in the helical component.

Intra-FCY1: a novel system to identify mutations that cause protein misfolding.

Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteins in vivo often rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novel in vivo system named Intra-FCY1 that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] in Saccharomyces cerevisiae. The Intra-FCY1 system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1 system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1 to illuminate the relationship between protein sequence and stability.

P. Ala278Val mutation might cause a pathogenic defect in HEXB folding leading to the Sandhoff disease.

Sandhoff disease is a rare neurodegenerative and autosomal recessive disorder, which is characterized by a defect in ganglioside metabolism. Also, it is caused by mutations in the HEXB gene for the β-subunit isoform 1 of β-N-acetyl hexosaminidase. In the present study, an Iranian 14- month -old girl with 8- month history of unsteady walking and involuntary movements was described. In this regard, biochemical testing showed some defects in the normal activity of beta-hexosaminidase protein. Following sequencing of HEXB gene, a homozygous c.833C > T mutation was identified in the patient's genome. After recognition of p.A278V, several different in silico methods were used to assess the mutant protein stability, ranging from mutation prediction methods to ligand docking. The p.A278V mutation might be disruptive because of changing the three-dimensional folding at the end of the 5th alpha helix. According to the medical prognosis, in silico and structural analyses, it was predicted to be disease cause.

Case report and literature review: A de novo pathogenic missense variant in ACTN4 gene caused rapid progression to end-stage renal disease

BackgroundFocal segmental glomerulosclerosis (FSGS) is a histopathological diagnosis of the sclerosis of glomeruli and the damage to renal podocytes. FSGS affects the filtration function of the kidneys and results in nephrotic syndrome (NS) in children and adults. FSGS is a clinically and genetically heterogeneous disorder. FSGS-1 [OMIM #603278] is one of the progressive hereditary renal diseases. It is caused by heterozygous variants of the actinin alpha 4 (ACTN4) [OMIM*604638] gene on chromosome 19q13.2 in a dominant inheritance (AD) manner. With the recent development of whole-exome sequencing (WES), 22 (including our case) pathogenic or likely pathogenic variants have been identified in ACTN4 gene.Case presentationWe reported a 17-year-old Chinese girl who was hospitalized with foamy urine, nausea and vomiting. Laboratory tests revealed increased levels of serum creatinine and urea nitrogen. Ultrasonography demonstrated bilaterally reduced size of kidneys. The primary diagnoses were NS and chronic kidney disease stage 5 (CKD5). The hemodialysis was initiated in 48 h after admission. After 4 months of treatment, the patient received an allogeneic kidney transplantation from her father. A novel heterozygous missense variant c.494C > T (p.A165V) in the ACTN4 gene was found by WES in the patient. This variant was confirmed by Sanger sequencing. The computational simulation of the stability of mutant protein (p.A165V) was decreased. Interatomic interactions of the p.A165V site were increased, and it might be associated with the increased ubiquitylation in the vicinity of the mutant site.ConclusionAs per the guidelines of the American College of Medical Genetics and Genomics for interpreting sequence variants, the novel heterozygous missense variant was pathogenic (PS2 + PM1 + PM2 + PP3 + PP4). It should be noted that the early onset of severe proteinuria with a poor prognosis is an important and universal symptom for most genetic FSGS. If necessary, genetic screening is recommended.

In-Silico Investigation of Effects of Single-Nucleotide Polymorphisms in PCOS-Associated CYP11A1 Gene on Mutated Proteins.

Polycystic ovary syndrome (PCOS) is a reproductive disorder with multiple etiologies, mainly characterized by the excess production of androgens. It is equally contributed to by genes and environment. The CYP11A1 gene is imperative for steroidogenesis, so any dysregulation or mutation in this gene can lead to PCOS pathogenesis. Therefore, nucleotide diversity in this gene can be helpful in spotting the likelihood of developing PCOS. The present study was initiated to investigate the effect of single nucleotide polymorphisms in human CYP11A1 gene on different attributes of encoded mutated proteins, i.e., sub-cellular localization, ontology, half-life, isoelectric point, instability index, aliphatic index, extinction coefficient, 3-D and 2-D structures, and transmembrane topology. For this purpose, initially coding sequence (CDS) and single nucleotide polymorphisms (SNPs) were retrieved for the desired gene from Ensembl followed by translation of CDS using EXPASY tool. The protein sequence obtained was subjected to different tools including CELLO2GO, ProtParam, PHYRE2, I-Mutant, SIFT, and PolyPhen. It was found that out of seventy-eight SNPs analyzed in this project, seventeen mutations, i.e., rs750026801 in exon 1, rs776056840, rs779154292 and rs1217014229 in exon 2, rs549043326 in exon 3, rs755186597 in exon 4, rs1224774813, rs757299093 and rs1555425667 in exon 5, rs1454328072 in exon 7, rs762412759 and rs755975808 in exon 8, and rs754610565, rs779413653, rs765916701, rs1368450780, and rs747901197 in exon 9 considerably altered the structure, sub-cellular localization, and physicochemical characteristics of mutated proteins. Among the fifty-nine missense SNPs documented in present study, fifty-five and fifty-three were found to be deleterious according to SIFT and PolyPhen tools, respectively. Forty-nine missense mutations were analyzed to have a decreasing effect on the stability of mutant proteins. Hence, these genetic variants can serve as potential biomarkers in human females for determining the probability of being predisposed to PCOS.

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding.

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.

Predicting the stability of mutant proteins by computational approaches: an overview.

A very large number of computational methods to predict the change in thermodynamic stability of proteins due to mutations have been developed during the last 30years, and many different web servers are currently available. Nevertheless, most of them suffer from severe drawbacks that decrease their general reliability and, consequently, their applicability to different goals such as protein engineering or the predictions of the effects of mutations in genetic diseases. In this review, we have summarized all the main approaches used to develop these tools, with a survey of the web servers currently available. Moreover, we have also reviewed the different assessments made during the years, in order to allow the reader to check directly the different performances of these tools, to select the one that best fits his/her needs, and to help naïve users in finding the best option for their needs.

Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

BackgroundNiemann-Pick type C (NP-C) is a rare neurovisceral genetic disorder caused by mutations in the NPC1 or the NPC2 gene. NPC1 is a multipass-transmembrane protein essential for egress of cholesterol from late endosomes/lysosomes. To evaluate impacts of NPC1 mutations, we examined fibroblast cultures from 26 NP-C1 patients with clinical phenotypes ranging from infantile to adult neurologic onset forms. The cells were tested with multiple assays including NPC1 mRNA expression levels and allele expression ratios, assessment of NPC1 promoter haplotypes, NPC1 protein levels, cellular cholesterol staining, localization of the mutant NPC1 proteins to lysosomes, and cholesterol/cholesteryl ester ratios. These results were correlated with phenotypes of the individual patients.ResultsOverall we identified 5 variant promoter haplotypes. Three of them showed reporter activity decreased down to 70% of the control sequence. None of the haplotypes were consistently associated with more severe clinical presentation of NP-C. Levels of transcripts carrying null NPC1 alleles were profoundly lower than levels of the missense variants. Low levels of the mutant NPC1 protein were identified in most samples. The protein localised to lysosomes in cultures expressing medium to normal NPC1 levels. Fibroblasts from patients with severe infantile phenotypes had higher cholesterol levels and higher cholesterol/cholesteryl ester ratios. On the contrary, cell lines from patients with juvenile and adolescent/adult phenotypes showed values comparable to controls.ConclusionNo single assay fully correlated with the disease severity. However, low residual levels of NPC1 protein and high cholesterol/cholesteryl ester ratios associated with severe disease. The results suggest not only low NPC1 expression due to non-sense mediated decay or low mutant protein stability, but also dysfunction of the stable mutant NPC1 as contributors to the intracellular lipid transport defect.

Homozygous STXBP1 variant causes encephalopathy and gain-of-function in synaptic transmission.

Heterozygous mutations in the STXBP1 gene encoding the presynaptic protein MUNC18-1 cause STXBP1 encephalopathy, characterized by developmental delay, intellectual disability and epilepsy. Impaired mutant protein stability leading to reduced synaptic transmission is considered the main underlying pathogenetic mechanism. Here, we report the first two cases carrying a homozygous STXBP1 mutation, where their heterozygous siblings and mother are asymptomatic. Both cases were diagnosed with Lennox-Gastaut syndrome. In Munc18-1 null mouse neurons, protein stability of the disease variant (L446F) is less dramatically affected than previously observed for heterozygous disease mutants. Neurons expressing Munc18L446F showed minor changes in morphology and synapse density. However, patch clamp recordings demonstrated that L446F causes a 2-fold increase in evoked synaptic transmission. Conversely, paired pulse plasticity was reduced and recovery after stimulus trains also. Spontaneous release frequency and amplitude, the readily releasable vesicle pool and the kinetics of short-term plasticity were all normal. Hence, the homozygous L446F mutation causes a gain-of-function phenotype regarding release probability and synaptic transmission while having less impact on protein levels than previously reported (heterozygous) mutations. These data show that STXBP1 mutations produce divergent cellular effects, resulting in different clinical features, while sharing the overarching encephalopathic phenotype (developmental delay, intellectual disability and epilepsy).

HSP90: Enabler of Cancer Adaptation

The stability and function of many oncogenic mutant proteins depend on heat shock protein 90 (HSP90). This unique activity has inspired the exploration of HSP90 as an anticancer target for over two decades. Unfortunately, while clinical trials of highly optimized HSP90 inhibitors have demonstrated modest benefit for patients with advanced cancers, most commonly stabilization of disease, no HSP90 inhibitor has demonstrated sufficient efficacy to achieve FDA approval to date. This review discusses potential reasons for the limited success of these agents and how our increasingly sophisticated understanding of HSP90 suggests alternative, potentially more effective strategies for targeting it to treat cancers. First, we focus on insights gained from model organisms that suggest a fundamental role for HSP90 in supporting the adaptability and heterogeneity of cancers, key factors underlying their ability to evolve and acquire drug resistance. Second, we examine how HSP90’s role in promoting the stability of mutant proteins might be targeted in genetically unstable tumor cells to reveal their aberrant, foreign proteome to the immune system. Both of these emerging aspects of HSP90 biology suggest that the most effective use of HSP90 inhibitors may not be at high doses with the intent to kill cancer cells, but rather in combination with other molecularly targeted therapies at modest, non-heat shock-inducing exposures that limit the adaptive capacity of cancers.

Variants identified in PTK7 associated with neural tube defects.

BackgroundVariants in planar cell polarity (PCP) pathway genes have been repeatedly implicated in the pathogenesis of NTDs in both mouse models and in human cohorts. Mouse models indicate that the homogenous disruption of the Ptk7 gene, a PCP regulator, results in craniorachischisis; while embryos that are doubly heterozygous for Ptk7XST87 and Vangl2 Lp mutations present with spina bifida.MethodsIn this study, we initially sequenced exons of the human PTK7 gene in 192 spina bifida patients and 190 controls from a California population. A phase II validation study was performed in 343 Chinese NTD cohort. Functional assays including immunoblotting and immunoprecipitation were used to study identified variants effect on PTK7 function.ResultsWe identified three rare (MAF <0.001) missense heterozygous PTK7 variants (NM_001270398.1:c.581C>T, p.Arg630Ser and p.Tyr725Phe) in the spina bifida patients. In our functional analyses, p.Arg630Ser affected PTK7 mutant protein stability and increased interaction with Dvl2, while the p.Thr186Met variant decreased PTK7 interactions with Dvl2. No novel predicted‐to‐be‐damaging variant or function‐disrupted PTK7 variant was identified among the control subjects. We subsequently re‐sequenced the PTK7 CDS region in 343 NTDs from China to validate the association between PTK7 and NTDs. The frequency of PTK7 rare missense variants in the Chinese NTD samples is significantly higher than in gnomAD controls.ConclusionOur study suggests that rare missense variants in PTK7 contribute to the genetic risk of NTDs.

Conformational dynamics of Ribonuclease Sa and its S48P mutant: Implications for the stability of the mutant protein

The optimization of β-turns has been used as a strategy to increase protein thermal stability. One example is the S48P mutation in Ribonuclease Sa, introduced to optimize a β-turn, which increases the stability of the protein as determined experimentally. Here, we have studied 48SYGY51 β-turn and its S48P mutant from RNase Sa, as a peptide and as part of the protein, using molecular dynamics simulations. The turn propensity of the region 48SYGY51 shows an increase in both the peptide and protein models on S48P mutation. The mutant protein shows an overall decrease in conformational dynamics and a decrease in conformational heterogeneity as compared to the wildtype protein. A comparatively restricted sampling of the φ-ψ region of GLN47, a pre PRO48 residue, in the mutant protein and some local changes in hydrogen bonding patterns involving residues 20–24 might be contributing to the mutant protein stability. In addition, some long-range hydrogen bonding interactions involving the 60s loop and the salt-bridge interaction involving ASP17-ARG63 could also be contributing to the increase in rigidity and stability of the mutant protein.

Computational analysis of non-synonymous single nucleotide polymorphism in the bovine cattle kappa-casein (CSN3) gene

Backgroundkappa-casein is candidate gene, present on chromosome 6 of cattle, play an important role in mammary gland and properties of milk. MethodsIn the present study, we used SIFT, SNAP2, PROVEAN and PolyPhen 2 computational tools which are based on different approaches to predict deleterious nsSNPs in coding region of cattle CSN3 gene. Furthermore, protein stability and structure were predicted by I-Mutant 2.0, MuStab, ModPred, ConSurf, PSIPRED, MUSTER, Ramachandran plot, TM-score, FT site and STRING. ResultsAmong the 15 nsSNPs, 3 were predicted deleterious by SIFT, SNAP2, PROVEAN and PolyPhen 2. Additionally, R118S, S125A and S176G variant showed decrease in stability, S125A and S176G are highly conserved and role in post translational modification using I-Mutant 2.0, MuStab, ConSurf and ModPred. The 3D structure of CSN3 wild type and mutant were generated by MUSTER and validated via Ramachandran plot, showed 170 residues (90.4%) in favoured region, 14 residues in allowing the region (7.4%) and 4 residues (2.2%) in outer region which indicated good quality model. The TM-score ranging from 0.70 to 0.87 which means two structures was within same fold while higher RMSD values (1.59 to 2.31) showed that the deviation from two structures may change the functional activity of the protein. Results of GROMOS96 indicated that the stability of mutant protein was decreased. Ligand binding site 1 has a SER-125, site 2, ASP-169 and site 3, SER-125. CSN3 protein interacts with 10 different proteins. ConclusionThe S125A variant of cattle population may serve as a genetic marker for mutational analysis of CSN3.

Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated With 46,XY Disorders of Sex Development in Egyptian Patients

BackgroundMutations in the HSD17B3 gene are associated with a 46,XY disorder of sexual development (46,XY DSD) as a result of low testosterone production during embryogenesis. AimTo elucidate the molecular basis of the disorder by chemically analyzing four missense mutations in HSD17B3 (T54A, M164T, L194P, G289S) from Egyptian patients with 46,XY DSD. MethodsExpression plasmids for wild-type 17β-hydroxysteroid hydrogenase type 3 (17β-HSD3) and mutant enzymes generated by site-directed mutagenesis were transiently transfected into human HEK-293 cells. Protein expression was verified by western blotting and activity was determined by measuring the conversion of radiolabeled Δ4-androstene-3,17-dione to testosterone. Application of a homology model provided an explanation for the observed effects of the mutations. OutcomesTestosterone formation by wild-type and mutant 17β-HSD3 enzymes was compared. ResultsMutations T54A and L194P, despite normal protein expression, completely abolished 17β-HSD3 activity, explaining their severe 46,XY DSD phenotype. Mutant M164T could still produce testosterone, albeit with significantly lower activity compared with wild-type 17β-HSD3, resulting in ambiguous genitalia or a microphallus at birth. The substitution G289S represented a polymorphism exhibiting comparable activity to wild-type 17β-HSD3. Sequencing of the SRD5A2 gene in three siblings bearing the HSD17B3 G289S polymorphism disclosed the homozygous Y91H mutation in the former gene, thus explaining the 46,XY DSD presentations. Molecular modeling analyses supported the biochemical observations and predicted a disruption of cofactor binding by mutations T54A and M164T and of substrate binding by L196P, resulting in the loss of enzyme activity. In contrast, the G289S substitution was predicted to disturb neither the three-dimensional structure nor enzyme activity. Clinical TranslationBiochemical analysis of mutant 17β-HSD3 enzymes is necessary to understand genotype-phenotype relationships. Strengths and LimitationsBiochemical analysis combined with molecular modeling provides insight into disease mechanism. However, the stability of mutant proteins in vivo cannot be predicted by this approach. ConclusionThe 17β-HSD3 G289S substitution, previously reported in other patients with 46,XY DSD, is a polymorphism that does not cause the disorder; thus, further sequence analysis was required and disclosed a mutation in SRD5A2, explaining the cause of 46,XY DSD in these patients.Engeli RT, Tsachaki M, Hassan HA, et al. Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated With 46,XY Disorders of Sex Development in Egyptian Patients. J Sex Med 2017;14:1165–1174.

In silico analysis of non-synonymous single nucleotide polymorphisms in human DAZL gene associated with male infertility

ABSTRACTIn living systems the most frequent type of genetic mutation is the single nucleotide polymorphism (SNP). Non-synonymous SNPs (nsSNPs) or missense mutations arise in coding regions of a particular gene. nsSNPs result in a single amino acid substitution which may have effects on the structure and/or function of proteins. Spermatogenesis is a complex process where haploid spermatozoa are formed. The deleted in azoospermia like (DAZL) gene has a relationship with male infertility and dysfunction of DAZL may decrease the sperm count which leads to oligozoospermia or azoospermia. Various computational methods were used to analyze the genetic variations of DAZL affecting the structure and/or function. In the present study, N109T was assigned as the most deleterious or disease related nsSNP by SIFT, MutPred, PolyPhen 2.0, I-Mutant, and MuStab tools. The ConSurf tool showed that functional amino acid residues which are conserved in Human DAZL include the N109T nsSNP. The secondary and tertiary structure was predicted using PSIPRED and MUSTER. Our study shows that the N109T variant may directly or indirectly weaken amino acid interactions and hydrogen bond networks of the DAZL protein, which we predicted may result in altered DAZL protein function. Further, computational analysis of free energy change due to this point mutation using GROMOS96 indicated decreased stability of the DAZL protein. The N109T variant in an infertile male population may provide a genetic marker for mutational analysis of DAZL.Abbreviations: DAZL: deleted in azoospermia like; dbSNP: database of single nucleotide polymorphism; nsSNPs: non-synonymous SNPs; AA: amino acid; SIFT: sorting intolerant from tolerant; PolyPhen-2: polymorphism phenotyping v2; MUSTER: multi-sources threader; PDB: protein data bank; MuStab: predicting mutant protein stability change; PSIPRED: PSI-blast based secondary structure prediction

Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease.

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.

Computational approaches for predicting mutant protein stability.

Mutations in the protein affect not only the structure of protein, but also its function and stability. Prediction of mutant protein stability with accuracy is desired for uncovering the molecular aspects of diseases and design of novel proteins. Many advanced computational approaches have been developed over the years, to predict the stability and function of a mutated protein. These approaches based on structure, sequence features and combined features (both structure and sequence features) provide reasonably accurate estimation of the impact of amino acid substitution on stability and function of protein. Recently, consensus tools have been developed by incorporating many tools together, which provide single window results for comparison purpose. In this review, a useful guide for the selection of tools that can be employed in predicting mutated proteins' stability and disease causing capability is provided.