Affect Protein Stability Research Articles

Cross-Ethnic Variant Screening and Burden Analysis of PTPA in Parkinson's Disease.

Recently, homozygous variants in PTPA were identified as the disease cause for two pedigrees with early-onset parkinsonism and intellectual disability. Although the initial link between PTPA and parkinsonism has been established, further replication was still necessary. To evaluate the genetic role of PTPA in Parkinson's disease (PD). We analyzed rare variants of PTPA in cohorts of Asian and European ancestries (Ncase = 2743, Ncontrol = 8177) with whole-exome sequencing, and further explored the functional effect of the target variant. One patient with early-onset PD from a consanguineous family carried the homozygous variant p.Met329Val, while her parents and elder sister with heterozygous p.Met329Val were healthy. This patient developed minor cognitive decline within 1 year, with a Montreal Cognitive Assessment (MoCA) score dropping from 28 to 25. Functional exploration with overexpression studies suggested that this variant was associated with decreased protein phosphatase 2A (PTPA) protein level by affecting protein stability, but not mRNA expression. These results have broadened the mutation spectrum of PTPA, and paved the way for further research into the role of PTPA in PD. © 2023 International Parkinson and Movement Disorder Society.

Associated Water Dynamics Might Be a Key Factor Affecting Protein Stability in the Crowded Milieu.

Over the past 20 years, the most studied and debated aspect of macromolecular crowding is how it affects protein stability. Traditionally, it is explained by a delicate balance between the stabilizing entropic effect and the stabilizing or destabilizing enthalpic effect. However, this traditional crowding theory cannot explain experimental observations like (i) negative entropic effect and (ii) entropy-enthalpy compensation. Herein, we provide experimental evidence that associated water dynamics plays a crucial role in controlling protein stability in the crowded milieu for the first time. We have correlated the modulation of associated water dynamics with the overall stability and its individual components. We showed that rigid associated water would stabilize the protein through entropy but destabilize it through enthalpy. In contrast, flexible associated water destabilizes the protein through entropy but stabilizes through enthalpy. Consideration of entropic and enthalpic modulation through crowder-induced distortion of associated water successfully explains the negative entropic part and entropy-enthalpy compensation. Furthermore, we argued that the relationship between the associated water structure and protein stability should be better understood by individual entropic and enthalpic components instead of the overall stability. Although a huge effort is necessary to generalize the mechanism, this report provides a unique way of understanding the relationship between protein stability and associated water dynamics, which might be a generic phenomenon and should trigger much research in this area.

Abstract 3544: ALK signaling activity stabilizes SLC3A2 protein levels in neuroblastoma tumorigenesis

Abstract Background: Neuroblastoma is the most common childhood tumor that grows in the adrenal glands or sympathetic ganglia. Approximately 10% of pediatric neuroblastoma patients harbor mutations in anaplastic lymphoma kinase (ALK). In a previous study, we identified SLC3A2 as a potential interacting partner with ALK using BioID-based in vivo proximity labeling. SLC3A2 is a multifunctional protein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and regulates polyamine transport. No previous link to ALK signaling has been reported and the underlying mechanisms and functional consequences of a SLC3A2-ALK interaction in ALK-driven neuroblastoma are unclear. Materials & Methods: The interaction of SLC3A2 and ALK initially detected with BioID-based in vivo proximity labeling was validated by immunoprecipitation. The effect of ALK signaling on SLC3A2 expression and protein stability was evaluated by quantitative PCR and immunoblotting. The functional effect of SLC3A2 on cell viability were investigated by siRNA and inhibitor treatments in both human and mouse ALK-driven neuroblastoma cells. Results: SLC3A2 was confirmed to interact with ALK in both anti-ALK and anti-SLC3A2 antibody co-immunoprecipitations from NB1 cells (ALK-wild-type) and CLB-GE (ALK-F1174V). Moreover, this interaction was decreased by ALK inhibitor (lorlatinib) treatment. Upon ALKAL2 ligand treatment, ALK signaling increased the protein levels of SLC3A2 and this was abrogated by lorlatinib treatment in NB1 cells. Furthermore, lorlatinib treatment suppressed SLC3A2 expression and significantly affected protein stability in neuroblastoma cells with different ALK mutations including CLB-BAR (ALK exon4-11 deletion), CLB-GAR (ALK-R1275Q), and CLB-GE (ALK-F1174V) during cycloheximide chase analysis. Knockdown of SLC3A2 significantly inhibited cell growth and down-regulated the amino acid transporter SLC7A5 (LAT1) expression in CLB-GE cells (ALK-F1174V). While mono treatment of either lorlatinib (ALK TKI) or AMXT-1501 (polyamine transport inhibitor) only showed moderate effects, combinatorial treatment exhibited a synergistic effect on cell growth in ALK-driven primary-cultured mouse neuroblastoma #9883 (Th-MYCN; Alk-F1178S) and #111 (Th-MYCN; Rosa26_Alkal2) cells. Conclusions: SLC3A2 protein stability and its interaction with ALK is dependent on ALK signaling in an ALK-driven neuroblastoma context. The synergistic effect of combined ALK and polyamine transport inhibitors suggests a potential therapeutic option for ALK-driven neuroblastoma. Citation Format: Wei-Yun Lai, Tzu-Po Chuang, Marcus Borenäs, Ruth Palmer, Bengt Hallberg. ALK signaling activity stabilizes SLC3A2 protein levels in neuroblastoma tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3544.

Abstract 1294: Cancer-associated missense mutations in the tumor suppressor Polybromo-1 variably affect protein stability and function

Abstract 1294: Cancer-associated missense mutations in the tumor suppressor Polybromo-1 variably affect protein stability and function

Site-specific methylation on α-N-terminus of peptides through chemical and enzymatic methods.

Protein α-N-terminal (Nα) methylation is a post-translational modification catalyzed by N-terminal methyltransferase 1/2 (NTMT1/2) and METTL13. Nα methylation affects protein stability, protein-protein interaction, and protein-DNA interaction. Thus, Nα methylated peptides are essential tools to study the function of Nα methylation, generate specific antibodies for different states of Nα methylation, and characterize the enzyme kinetics and activity. Here, we describe chemical methods of site-specific synthesis of Nα mono-, di-, and trimethylated peptides in the solid phase. In addition, we describethe preparation of trimethylation peptides by recombinant NTMT1 catalysis.

Deciphering Molecular Factors That Affect Electron Transfer at the Cell Surface of Electroactive Bacteria: The Case of OmcA from Shewanella oneidensis MR-1

Multiheme cytochromes play a central role in extracellular electron transfer, a process that allows microorganisms to sustain their metabolism with external electron acceptors or donors. In Shewanella oneidensis MR-1, the decaheme cytochromes OmcA and MtrC show functional specificity for interaction with soluble and insoluble redox partners. In this work, the capacity of extracellular electron transfer by mutant variants of S. oneidensis MR-1 OmcA was investigated. The results show that amino acid mutations can affect protein stability and alter the redox properties of the protein, without affecting the ability to perform extracellular electron transfer to methyl orange dye or a poised electrode. The results also show that there is a good correlation between the reduction of the dye and the current generated at the electrode for most but not all mutants. This observation opens the door for investigations of the molecular mechanisms of interaction with different electron acceptors to tailor these surface exposed cytochromes towards specific bio-based applications.

The mitochondrial micropeptide Stmp1 promotes retinal cell differentiation

During mammalian retinal development, the differentiation of multipotent progenitors depends on the coordinated action of a variety of intrinsic factors including non-coding RNAs (ncRNAs). To date, many small open reading frames have been identified in ncRNAs to encode micropeptides that function in diverse biological processes; however, it remains unclear whether they have a role in retinal development. Here we report that the 47-amino acid (AA) mitochondrial micropeptide Stmp1 encoded by the lncRNA 1810058I24Rik is involved in retinal differentiation. As the major protein product of 1810058I24Rik, Stmp1 promotes the differentiation of bipolar, amacrine and Müller cells as 1810058I24Rik does when overexpressed in neonatal murine retinas. Moreover, we have identified the 15-AA N-terminus of Stmp1 as its mitochondrion-targeting sequence as well as 5 conserved AA residues that affect protein stability and/or retinal cell differentiation. Together, our data reveal several novel characteristics of Stmp1 and uncover a role for Stmp1 in retinal cell differentiation perhaps through regulating mitochondrial function.

Characterization of the Escherichia coli pyridoxal 5'-phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability.

The pyridoxal 5′‐phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP‐dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP‐dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.

Impact of the Cellular Environment on Adenosine Triphosphate Conformations.

The cytoplasm is an environment crowded by macromolecules and filled with metabolites and ions. Recent experimental and computational studies have addressed how this environment affects protein stability, folding kinetics, and protein-protein and protein-nucleic acid interactions, though its impact on metabolites remains largely unknown. Here we show how a simulated cytoplasm affects the conformation of adenosine triphosphate (ATP), a key energy source and regulatory metabolite present at high concentrations in cells. Analysis of our all-atom model of a small volume of the Escherichia coli cytoplasm when contrasted with ATP modeled in vitro or resolved with protein structures deposited in the Protein Data Bank reveals that ATP molecules bound to proteins in cell form specific pitched conformations that are not observed at significant concentrations in the other environments. We hypothesize that these interactions evolved to fulfill functional roles when ATP interacts with protein surfaces.

IQSEC2-related encephalopathy in male children: Novel mutations and phenotypes.

The isoleucine–glutamine (IQ) motif and Sec7 domain-containing protein 2 (IQSEC2) gene, located at Xp11. 2, are associated with nervous system diseases, such as epilepsy, autism, and intellectual disabilities. Gender-related differences in the severity of phenotype severity have been described previously. Here, we report the details of seven male children with IQSEC2 mutations from different families. During this investigation, we explored the relationship between the genotype and phenotype of IQSEC2 mutations; to do so, we recruited seven children with pathogenic/likely pathogenic IQSEC2 mutations who were diagnosed with global developmental delay and/or epilepsy. Their clinical features were assessed, and Trio-based whole-exome sequencing (trio WES) was conducted in seven pedigrees. A variety of algorithms and computational tools were used to calculate the pathogenicity, protein stability, conservation, side chain properties, and protein-protein interactions of mutated proteins. The seven patients ranged in age from 18 months to 5 years. Among them, six children were found to have both developmental delay and epilepsy, and one child only exhibited developmental delay. Four novel mutations (c.316C > T, c.443_4 44dup, c.3235T > C, and c.1417G > T) were newly reported. Two patients did not have truncated aberrant proteins caused by missense mutations. Still, they did have severe phenotypes, such as early-onset epilepsy in infancy, because the mutations were located in domains like the pleckstrin homology and IQ calmodulin-binding motif domains. The bioinformatics analysis also proved that missense mutations may be located in the functional region, which affects protein stability and is harmful. In summary, severe phenotypes, such as early-onset epilepsy in infancy, occur in male patients with a missense mutation in specific domains (e.g., pleckstrin homology and IQ calmodulin-binding motif domains). Some female individuals with IQSEC2 mutations may be asymptomatic because of the skewed inactivation of the X chromosome.

Molecular and Structural Analysis of Specific Mutations from Saudi Isolates of SARS-CoV-2 RNA-Dependent RNA Polymerase and their Implications on Protein Structure and Drug-Protein Binding.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has stressed the global health system to a significant level, which has not only resulted in high morbidity and mortality but also poses a threat for future pandemics. This situation warrants efforts to develop novel therapeutics to manage SARS-CoV-2 in specific and other emerging viruses in general. This study focuses on SARS-CoV2 RNA-dependent RNA polymerase (RdRp) mutations collected from Saudi Arabia and their impact on protein structure and function. The Saudi SARS-CoV-2 RdRp sequences were compared with the reference Wuhan, China RdRp using a variety of computational and biophysics-based approaches. The results revealed that three mutations—A97V, P323I and Y606C—may affect protein stability, and hence the relationship of protein structure to function. The apo wild RdRp is more dynamically stable with compact secondary structure elements compared to the mutants. Further, the wild type showed stable conformational dynamics and interaction network to remdesivir. The net binding energy of wild-type RdRp with remdesivir is -50.76 kcal/mol, which is more stable than the mutants. The findings of the current study might deliver useful information regarding therapeutic development against the mutant RdRp, which may further furnish our understanding of SARS-CoV-2 biology.

R17C Mutation in Photoreceptor Disc-Specific Protein, PRCD, Results in Additional Lipidation Altering Protein Stability and Subcellular Localization.

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein essential for maintaining OS structures while contributing to rhodopsin packaging densities and distribution in disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation linked with retinitis pigmentosa (RP) in humans and dogs demonstrates the importance of palmitoylation for protein stability and trafficking to the OS. We demonstrate a mutation, in the polybasic region (PBR) of PRCD (Arg17Cys) linked with RP where an additional lipidation is observed through acyl-RAC. Immunolocalization of transiently expressed R17C in hRPE1 cells depicts similar characteristics to wild-type PRCD; however, a double mutant lacking endogenous palmitoylation at Cys2Tyr with Arg17Cys is comparable to the C2Y protein as both aggregate, mislocalized to the subcellular compartments within the cytoplasm. Subretinal injection of PRCD mutant constructs followed by electroporation in murine retina exhibit mislocalization in the inner segment. Despite being additionally lipidated and demonstrating strong membrane association, the mutation in the PBR affects protein stability and localization to the OS. Acylation within the PBR alone neither compensates for protein stability nor trafficking, revealing defects in the PBR likely lead to dysregulation of PRCD protein associated with blinding diseases.

In-silico investigation of systematic missense mutations of middle east respiratory coronavirus spike protein.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe pneumonia-like symptoms and is still pose a significant threat to global public health. A key component in the virulence of MERS-CoV is the Spike (S) protein, which binds with the host membrane receptor dipeptidyl peptidase 4 (DPP4). The goal of the present investigation is to examine the effects of missense mutations in the MERS-CoV S protein on protein stability and binding affinity with DPP4 to provide insight that is useful in developing vaccines to prevent coronavirus infection. We utilized a saturation mutagenesis approach to simulate all possible mutations in the MERS-CoV full-length S, S Receptor Binding Domain (RBD) and DPP4. We found the mutations in MERS-CoV S protein residues, G552, C503, C526, N468, G570, S532, S451, S419, S465, and S435, affect protein stability. We identified key residues, G538, E513, V555, S557, L506, L507, R511, M452, D537, and S454 in the S protein RBD region are important in the binding of MERS-CoV S protein to the DPP4 receptor. We investigated the effects of MERS-CoV S protein viral mutations on protein stability and binding affinity. In addition, we studied all DPP4 mutations and found the functional substitution R336T weakens both DPP4 protein stability and S-DPP4 binding affinity. We compared the S protein structures of MERS-CoV, SARS-CoV, and SARS-CoV-2 viruses and identified the residues like C526, C383, and N468 located in equivalent positions of these viruses have effects on S protein structure. These findings provide further information on how mutations in coronavirus S proteins effect protein function.

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Importance of the Q/N-rich segment for protein stability of endogenous mouse TDP-43

TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). The long-disordered C-terminal region (CTR) of TDP-43 is known to be aggregation-prone and a hotspot for ALS mutations, so elucidation of the physiological function of CTR will provide insights into the pathogenesis of ALS. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch (HP) and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the embryonic lethality of homozygotes, and protein and mRNA expression levels of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of the Q/N-rich segment greatly restored the protein stability of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase–related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid–containing peptides and other periplasmic biomolecules in manifold pathways.

Sample Processing Considerations for Protein Stability Studies of Low Concentration Biofluid Samples using Differential Scanning Calorimetry.

The analysis of biofluid samples with low protein content (e.g., urine or saliva) can be challenging for downstream analysis methods with limited sensitivity. To circumvent this problem, sample processing methods are employed to increase the protein concentration in analyzed samples. However, for some techniques, like differential scanning calorimetry (DSC) that characterizes thermally-induced unfolding of biomolecules, sample processing must not affect native protein structure and stability. We evaluated centrifugal concentration and stirred cell ultrafiltration, two common methods of sample concentration characterized by a low risk of protein denaturation, with the goal of establishing a protocol for DSC analysis of low concentration biospecimens. Our studies indicate that both methods can affect protein stability assessed by DSC and, even after optimization of several parameters, the obtained DSC profile (thermogram) suggested that sample processing affects the structure or intermolecular interactions of component proteins contributing to altered thermal stability detectable by DSC. We also found a relationship between changes in thermograms and low protein concentration, indicating that diluting biospecimens to concentrations below 0.1 mg/mL can perturb the intermolecular environment and affect the structure of proteins present in the solution. Dilution of samples below 0.1 mg/mL, as well as concentration of samples with low protein content, resulted in affected thermogram shapes suggesting changes in protein stability. This should be taken into account when concentrating dilute samples or employing techniques that lower the protein concentration (e.g., fractionation), when downstream applications include techniques, such as DSC, that require the preservation of native protein forms.

Detection of novel variant and functional study in a Chinese family with nonsyndromic oligodontia.

To investigate the pathogenic gene of a patient with nonsyndromic oligodontia, and analyze its possible pathogenic mechanism. The variant was detected by whole exome sequencing (WES) and Sanger sequencing in a family with oligodontia. Bioinformatic and structural analyses were used to analyze variant. Functional studies including western blotting and immunofluorescent analyses and luciferase reporter assay were conducted to explore the functional effects. We identified a novel frameshift variant of PAX9 (c.491-510delGCCCT-ATCACGGCGGCGGCC, p.P165Qfs*145) outside the DNA-binding domain causing an autosomal-dominant nonsyndromic oligodontia in a Chinese family. Bioinformatic and structural analyses revealed that the variant is pathogenic and conserved evolutionarily, and the changes might affect protein stability or folding. Functional studies demonstrate dramatically reduced ability in activating transcription activity of BMP4 promoter and a marked decrease in protein production, as evaluated by western blotting and immunofluorescent analyses. We found a novel frameshift variant of PAX9 causing nonsyndromic oligodontia in a Chinese family. Our findings indicate that frameshift variants cause loss of function of PAX9 protein during the patterning of the dentition and the subsequent tooth agenesis, providing new molecular insights into the role of frameshift variant of PAX9 and broaden the pathogenic spectrum of PAX9 variants.

The Genetic and Molecular Analyses of RAD51C and RAD51D Identifies Rare Variants Implicated in Hereditary Ovarian Cancer from a Genetically Unique Population.

Simple SummaryWe have investigated RAD51C and RAD51D, hereditary ovarian cancer risk genes, in French Canadians of Quebec, Canada. This population of Western European origins exhibits a unique genetic landscape as shown by the frequency of carriers of specific rare pathogenic variants. As studying French Canadians could facilitate the identification and interpretation of clinically relevant variants, we performed genetic analyses of RAD51C and RAD51D in this population comprised of cases with a family history of ovarian cancer or those who developed it at a young age. We identified candidate variants and then investigated them in other French Canadian study groups. We performed biological assays and revealed possible mechanisms that would affect gene function. Using engineered cells expressing one of our protein variants, we also showed that they were more sensitive to a recently approved treatment for ovarian cancer. Our findings support the role of inherited variants in RAD51C and RAD51D in ovarian cancer.To identify candidate variants in RAD51C and RAD51D ovarian cancer (OC) predisposing genes by investigating French Canadians (FC) exhibiting unique genetic architecture. Candidates were identified by whole exome sequencing analysis of 17 OC families and 53 early-onset OC cases. Carrier frequencies were determined by the genetic analysis of 100 OC or HBOC families, 438 sporadic OC cases and 1025 controls. Variants of unknown function were assayed for their biological impact and/or cellular sensitivity to olaparib. RAD51C c.414G>C;p.Leu138Phe and c.705G>T;p.Lys235Asn and RAD51D c.137C>G;p.Ser46Cys, c.620C>T;p.Ser207Leu and c.694C>T;p.Arg232Ter were identified in 17.6% of families and 11.3% of early-onset cases. The highest carrier frequency was observed in OC families (1/44, 2.3%) and sporadic cases (15/438, 3.4%) harbouring RAD51D c.620C>T versus controls (1/1025, 0.1%). Carriers of c.620C>T (n = 7), c.705G>T (n = 2) and c.137C>G (n = 1) were identified in another 538 FC OC cases. RAD51C c.705G>T affected splicing by skipping exon four, while RAD51D p.Ser46Cys affected protein stability and conferred olaparib sensitivity. Genetic and functional assays implicate RAD51C c.705G>T and RAD51D c.137C>G as likely pathogenic variants in OC. The high carrier frequency of RAD51D c.620C>T in FC OC cases validates previous findings. Our findings further support the role of RAD51C and RAD51D in hereditary OC.

In Silico Tools for Analysis of Single-Nucleotide Polymorphisms in the Bovine Transferrin Gene.

Simple SummarySubclinical mastitis represents a significant disease in dairy animals and negatively impacts udder health. Genetics, pathogens, and poor health and management procedures in dairy animals contribute primarily to this disease. Animal genetics plays a major role in disease resistance and susceptibility. Studying single-nucleotide polymorphisms in genes helps to detect resistant and susceptible animals. Transferrin is one of the important glycoproteins involved in the innate immune system that prevents iron availability to pathogens. Several studies have reported polymorphism in the transferrin gene in dairy animals with mastitis, but no detailed information on in silico tools is available for identification and verification. So, this study was carried out to detect polymorphism in the transferrin gene in healthy and mastitic cows, and to use various in silico based tools and software to find the impact of single-nucleotide polymorphisms (SNPs) on animal health. The results indicated three SNPs were located in the transferrin gene of Holstein Friesian cross-bred dairy cows. Moreover, the use of online computational tools provided significant knowledge regarding whether the SNPs would be deleterious, damaging, benign, neutral, or affect protein stability and function.Dairy cattle with a high milk yield are susceptible to many infectious diseases, such as mastitis. Subclinical mastitis (SCM) is the most prevalent form of mastitis that predominantly affects animal health, and causes adverse effects on the quality and quantity of milk. In dairy animals, subclinical mastitis often remains undetected, as no gross changes in udder characteristics are visible. In the present study, 135 Holstein Friesian dairy animals were selected and screened as healthy (n = 25) and mastitic (n = 110) based on diagnostic tests such as the California mastitis test, pH, electrical conductivity, and somatic cell count. In this study, the somatic cell count was used as a gold-standard test in differentiating subclinical mastitis animals from healthy ones. The present study was carried out to study polymorphisms in the bovine transferrin gene in cows (with subclinical mastitis and healthy). For the early detection of resistant/or susceptible animals, a useful marker could be provided by the detection of single-nucleotide polymorphisms (SNPs) in the transferrin gene, which are often associated with mammary innate immune response. The sequencing results revealed three nucleotide substitutions: two transversions (230 A > C, 231 C > A) and one transition (294 A > G) in susceptible cows as compared to disease-free subjects. The nucleotide variations at position 230 (GAC > GCA) and 231 (GAC > GCA) were nonsynonymous, and corresponded to an amino acid change from aspartic acid to alanine; whereas at position 294 (GAA > GAG), the mutation was synonymous. In the present study, many in silico tools were taken into consideration to determine the effect of SNPs on protein structure and function. The PROVEAN tool found the amino acid substitution to be neutral and deleterious. PolyPhen-2 revealed the amino acid variations at positions 320 and 321 to most likely be damaging; and at the 341 position, the variations were benign. The I-Mutant and MUpro tools found that the protein stability decreased for nonsynonymous variations. The SIFT tool revealed the protein function was likely to be affected in nonsynonymous variations, with no change in the case of synonymous ones. Phylogenetic analysis of the bovine transferrin gene revealed a close relation of the CA allele with the Bos taurus transferrin, while the G allele was closely related to a cross of Bos indicus × Bos taurus serotransferrins, followed by the Bison bison transferrin. The least relation was shown by both alleles to Capra hircus, Ovis aries, and Bubalus bubalis.