Structure Of Mutant Protein Research Articles

Extrapolating the effect of non-synonymous SNP in bread wheat HSP16.9B gene: a molecular modelling and dynamics study

Small heat shock proteins (sHSP) are molecular chaperons which play a key role in protein homeostasis under stress conditions. Point mutation of aspartic acid (D) substitution for asparagine (N) at residue 11 (D11N) in HSP16.9B protein was predicted in HSP16.9B gene in wheat. However, its impact on protein function and structural consequences has not been explored. In this study, we examined the effect of point mutation using molecular modelling and molecular dynamics (MD) simulations. Moreover, point mutation induced addition of beta-sheet before the mutation position in the mutant protein. Three-dimensional homology protein modelling, structure validation, and molecular dynamics were carried out to investigate the conformational transitions and dynamics of the HSP16.9B protein due to D11N non-synonymous single nucleotide polymorphism (nsSNP). The MD results indicated that the stability of the mutant protein structure during entire simulation runs. Altogether, our investigation will provide useful understanding related to structural and functional basis of HSP16.9.

An in vivo and in silico analysis of novel variation in TMBIM6 gene affecting cardiopulmonary traits of Indian goats.

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) gene acts as calcium leak channel and negatively regulates autophagy and autophagosome formation. The TMBIM6 gene was amplified and searched for variation in three different goat populations (i.e. Black Bengal, Ganjam and Raighar) of Odisha state of the India. The result indicated two substitutions i.e. 55th position (C55T) and 95th position (C95A) in the amplified region of the gene resulting in change of amino acids (Leu>Phe and Thr>Asn). The identified SNPs were combined to form haplotypes and animals were grouped accordingly. Structural analysis showed minor changes (5%) in between mutant and wild TMBIM6 protein structures. However, any functional variation could not be identified with respect to the calcium ligand and open pore state. But an alteration of calcium binding site was found. The binding interaction of calcium with the TMBIM6 protein was hydrophobic in nature in closed state whereas hydrophilic in open pore stage. The stress releasing function was the result of calcium leakage controlled by amino acids coded by exon 4 and exon 5 regions of TMBIM6 gene. The effect of breed and haplotype on cardiopulmonary traits was studied. The data on cardiopulmonary traits of body i.e. rectal temperature, skin temperature, heart rate and respiration rate were recorded when ambient temperature usually remained the highest. The statistical analysis showed, significant difference in rectal temperature, skin temperature and respiration rate among these goat populations. The haplotypes (CC and TA) were found to have a significant (P<0.05) effect on rectal temperature, skin temperature and respiration rate. However, any such significant effect could not be identified in recorded heart rate. The objective of the present study to identify the genetic variations in TMBIM6 gene having significant effect on cardiopulmonary traits which can be further uses as the molecular markers to improve heat tolerance mechanism in goats.

Prediction of Deleterious Non-synonymous SNPs of Human STK11 Gene by Combining Algorithms, Molecular Docking, and Molecular Dynamics Simulation.

Serine-threonine kinase11 (STK11) is a tumor suppressor gene which plays a key role in regulating cell growth and apoptosis. It is widely known as a multitasking kinase and engaged in cell polarity, cell cycle arrest, chromatin remodeling, energy metabolism, and Wnt signaling. The substitutions of single amino acids in highly conserved regions of the STK11 protein are associated with Peutz–Jeghers syndrome (PJS), which is an autosomal dominant inherited disorder. The abnormal function of the STK11 protein is still not well understood. In this study, we classified disease susceptible single nucleotide polymorphisms (SNPs) in STK11 by using different computational algorithms. We identified the deleterious nsSNPs, constructed mutant protein structures, and evaluated the impact of mutation by employing molecular docking and molecular dynamics analysis. Our results show that W239R and W308C variants are likely to be highly deleterious mutations found in the catalytic kinase domain, which may destabilize structure and disrupt the activation of the STK11 protein as well as reduce its catalytic efficiency. The W239R mutant is likely to have a greater impact on destabilizing the protein structure compared to the W308C mutant. In conclusion, these mutants can help to further realize the large pool of disease susceptibilities linked with catalytic kinase domain activation of STK11 and assist to develop an effective drug for associated diseases.

Identification of insulin gene variants in patients with neonatal diabetes in the Chinese population.

Aims/IntroductionNeonatal diabetes mellitus is created by alterations in the genes responsible for beta‐cell mass and/or function. The present study aimed to evaluate the genetic variants in the insulin gene (INS) in four Chinese infants aged <12 months with diabetes onset, and to explore the clinical and genetic characteristics of permanent neonatal diabetes mellitus caused by INS mutations.Materials and MethodsThe complete coding sequences of KCNJ11, ABCC8 and INS were detected using Sanger sequencing. The pathogenicity of the mutations was determined based on the American College of Medical Genetics and Genomics, and the structure of wild‐type and mutant proteins was predicted using the web‐based tool, Phyre2.ResultsOne novel mutation (p.I99_C100insSI) and three previously reported variants (p.G32S, p.R89C and p.C96R) in INS were identified in four infants with early‐onset diabetes. All the mutations in the four patients were de novo. Except for mutation R89C, which causes permanent neonatal diabetes mellitus through the addition of an additional cysteine residue at the cleavage site of the A chain and C‐peptide, the other three mutations affected disulfide bonds. The patients had diabetes with marked hyperglycemia or diabetic ketoacidosis, and were then treated with exogenous insulin. Mutations in crucial regions of the INS might give rise to diabetes with varying severity.ConclusionsThis study enriches our awareness of the mutant spectrum in INS, and suggests the important role of INS in the development of permanent neonatal diabetes mellitus.

Computational analysis of functional SNPs in Alzheimer's disease-associated endocytosis genes.

BackgroundFrom genome wide association studies on Alzheimer’s disease (AD), it has been shown that many single nucleotide polymorphisms (SNPs) of genes of different pathways affect the disease risk. One of the pathways is endocytosis, and variants in these genes may affect their functions in amyloid precursor protein (APP) trafficking, amyloid-beta (Aβ) production as well as its clearance in the brain. This study uses computational methods to predict the effect of novel SNPs, including untranslated region (UTR) variants, splice site variants, synonymous SNPs (sSNPs) and non-synonymous SNPs (nsSNPs) in three endocytosis genes associated with AD, namely PICALM, SYNJ1 and SH3KBP1.Materials and MethodsAll the variants’ information was retrieved from the Ensembl genome database, and then different variation prediction analyses were performed. UTRScan was used to predict UTR variants while MaxEntScan was used to predict splice site variants. Meta-analysis by PredictSNP2 was used to predict sSNPs. Parallel prediction analyses by five different software packages including SIFT, PolyPhen-2, Mutation Assessor, I-Mutant2.0 and SNPs&GO were used to predict the effects of nsSNPs. The level of evolutionary conservation of deleterious nsSNPs was further analyzed using ConSurf server. Mutant protein structures of deleterious nsSNPs were modelled and refined using SPARKS-X and ModRefiner for structural comparison.ResultsA total of 56 deleterious variants were identified in this study, including 12 UTR variants, 18 splice site variants, eight sSNPs and 18 nsSNPs. Among these 56 deleterious variants, seven variants were also identified in the Alzheimer’s Disease Sequencing Project (ADSP), Alzheimer’s Disease Neuroimaging Initiative (ADNI) and Mount Sinai Brain Bank (MSBB) studies.DiscussionThe 56 deleterious variants were predicted to affect the regulation of gene expression, or have functional impacts on these three endocytosis genes and their gene products. The deleterious variants in these genes are expected to affect their cellular function in endocytosis and may be implicated in the pathogenesis of AD as well. The biological consequences of these deleterious variants and their potential impacts on the disease risks could be further validated experimentally and may be useful for gene-disease association study.

Conserved Arg451 residue is critical for maintaining the stability and activity of thioredoxin glutathione reductase

Thioredoxin glutathione reductase (TGR), a potential anthelminthic drug target causes NADPH-dependent transfer of electrons to both thioredoxins and glutathione systems. In the present study, we showed that a single point mutation conserved at Arg451 position is critical for maintaining the structure-function of FgTGR. The current biochemical results showed that R451A mutation significantly decreases both oxidoreductase activities (glutathione reductase and thioredoxin reductase) of the enzyme. Computational analyses using molecular dynamics simulation provided an in-depth insight into the structural alterations caused as a result of the mutation. Furthermore, the different regions of the mutant FgTGR structure were found to be altered in flexibility/rigidity as a result of the mutation. This led to mutant-specific conformational alterations and dominant differential motions that contributed to the abrogated function of mutant FgTGR. These results were confirmed using GdnHCl-induced denaturation-based stability studies. Moreover, mutation reduced the free energy of stabilization of the protein, thereby destabilizing the mutant protein structure. Therefore, these findings displayed differential dynamics in the FgTGR structure and highlighted the relevance of residue-level interactions in the protein. Thus, the current study provided a basis for exploiting regions other than the active site of TGR for inhibitory effect and development of novel antihelminthics.

Structural and Functional Analysis of human lung cancer risk associated hOGG1 variant Ser326Cys in DNA repair gene by molecular dynamics simulation.

Oxidative damaged DNA base lesions are repaired through human 8-oxoguanine DNA glycosylase gene (hOGG1) mediated pathways. A recent report based on the meta-analysis has suggested that the DNA Repair Gene hOGG1 variant Ser326Cys [3p26.2; allele S/C in nucleotide position αHelix2 Ser⇒Cys326] was associated with Lung Cancer risk in Caucasian population will alter the level Zhong et al., 2012. To the best of our knowledge, there has not been any such comprehensive in-silico investigation that validates the functional and structural impact of non-synonymous Lung Cancer Risk Associated Protein Domain (LCRAPD) mutation Ser326Cys (rs1052133) by molecular dynamics (MD) simulation approach following prediction of hOGG1 protein before and after the mutation. Further to the native and mutant protein structures, the amino acid residue and its secondary structure were observed through a solvent accessibility model for protein stability confirmation at the point of mutation. Taken together, this study suggests that the protein functional and structural studies could be a reasonable approach for investigating the impact of nsSNPs in future studies. In addition, 4295 patients samples incorporate with the analysis that genomic data types from cBioPortal. In the result, 4295 cases (91.5%) had alterations in all genes but the frequency of alterations in our targeted hOGG1 gene was shown with and without case alteration in the ratio (Logrank Test P-Value: 0.670) Kaplan-Meier by the number of patients at risk of the survival function.

Structural insight into conformational dynamics of non-active site mutations in KasA: A Mycobacterium tuberculosis target protein

The enzyme β-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115–147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb.

Identification of a missense variant in CLDN2 in obstructive azoospermia.

Obstructive azoospermia (OA), defined as an obstruction in any region of the male genital tract, accounts for 40% of all azoospermia cases. Of all OA cases, ~30% are thought to have a genetic origin, however, hitherto, the underlying genetic etiology of the majority of these cases remain unknown. To address this, we took a family-based whole-exome sequencing approach to identify causal variants of OA in a multiplex family with epidydimal obstruction. A novel gain-of-function missense variant in CLDN2 (c.481G>C; p.Gly161Arg) was found to co-segregate with the phenotype, consistent with the X-linked inheritance pattern observed in the pedigree. To assess the pathogenicity of this variant, the wild and mutant protein structures were modeled and their potential for strand formation in multimeric form was assessed and compared. The results showed that dimeric and tetrameric arrangements of Claudin-2 were not only reduced, but were also significantly altered by this single residue change. We, therefore, envisage that this amino acid change likely forms a polymeric discontinuous strand, which may lead to the disruption of tight junctions among epithelial cells. This missense variant is thus likely to be responsible for the disruption of the blood-epididymis barrier, causing dislodged epithelial cells to clog the genital tract, hence causing OA. This study not only sheds light on the underlying pathobiology of OA, but also provides a basis for more efficient diagnosis in the clinical setting.

Genotype-phenotype correlation of gangliosidosis mutations using in silico tools and homology modeling

Gangliosidoses, including GM1-gangliosidosis and GM2-gangliosidosis (Tay-Sachs disease and Sandhoff disease), are lysosomal disorders resulting from enzyme deficiencies and accumulation of gangliosides. Phenotypes of gangliosidoses range from infantile, late-infantile, juvenile, and to the adult form. The genotype-phenotype correlation is essential for prognosis and clinical care planning for patients with a gangliosidosis condition. Previously, we have developed a method to establish the genotype-phenotype correlation of another lysosomal disease, mucopolysaccharidosis type I, with in silico tools. This same method was applied to analyze the genotype and phenotype of 38 patients diagnosed with a gangliosidosis disease in the United States. Out of 40 mutations identified, 3 were novel, including p.Tyr192His and p.Phe556Ser of the GLB1 gene and p.Gly461Val of the HEXA gene. Furthermore, the mutant protein structure of all missense mutations was constructed by homology modeling. A systemic structural analysis of these models revealed the specific mechanisms of how each mutation may lead to the disease. In summary, the method developed in this study holds promise as a tool that can be broadly applicable to other lysosomal diseases and monogenic diseases.

Anticancer Screening of the Phytochemicals Present in the Medicinal Plant Vitex Negundo Against Mutant Anaplastic Lymphoma Kinase (ALK) Protein: An In-Silico Approach

The lymphomas are a heterogeneous group of cancer of the lymphocytes and the lymphatic system and accounts for up to 3% of all malignancies.1 Most of the drugs currently used for the treatment of lymphoma produce various side effects, hence in this study, we focus on natural compounds, obtained from the medicinal plant Vitex negundo, which exhibits selective toxicity against cancer cells. The objective of this research was to formulate the binding energies and interaction of selected phytochemicals present in the medicinal plant Vitex negundo2 against anaplastic lymphoma kinase protein, which is overexpressed in an anaplastic large cell lymphoma.3, 4,5 The structure of mutant human anaplastic lymphoma kinase protein was retrieved from the Protein Data Bank (PDB ID:4ANL ) and the 3D chemical structure of the phytochemicals present in the medicinal plant Vitex negundo was obtained from the PubChem database. Molecular docking study was performed for these natural compounds to evaluate and analyze their anti-lymphoma-cancer activity. A total of 16 compounds present in Vitex negundo, based on a comprehensive literature survey was selected for this molecular screening. Molecular docking analysis was carried out by Molegro Virtual Docker software, to screen the 16 chosen compounds and rank them according to their binding affinity towards the site of interaction of the oncoprotein, anaplastic lymphoma kinase. Out of the 16 screened phytocompounds, only 4 compounds showed promising interactions against the oncoprotein ALK (4ANL). 6’-p-hydroxybenzoyl mussaenosidic acid exhibited a very good binding with a molecular docking score of -127.723 kcal/mol, ranking first among the compounds screened. This was followed by Betulinic acid, Viridiflorol and protocatechuic acid with molecular docking scores of -95.596 kcal/mol, -76.1648 kcal/mol and -63.0854 kcal/mol and - respectively. The docking scores from the above study shows that the phytocompounds present in Vitex negundo extract exhibits an effective inhibitory effect against anaplastic lymphoma kinase protein that is over expressed in lymphoma.

Computational Screening and Analysis of Lung Cancer Related Non-Synonymous Single Nucleotide Polymorphisms on the Human Kirsten Rat Sarcoma Gene.

The human KRAS (Kirsten rat sarcoma) is an oncogene, involved in the regulation of cell growth and division. The mutations in the KRAS gene have the potential to cause normal cells to become cancerous in human lungs. In the present study, we focus on non-synonymous single nucleotide polymorphisms (nsSNPs), which are point mutations in the DNA sequence leading to the amino acid variants in the encoded protein. To begin with, we developed a pipeline to utilize a set of computational tools in order to obtain the most deleterious nsSNPs (Q22K, Q61P, and Q61R) associated with lung cancer in the human KRAS gene. Furthermore, molecular dynamics simulation and structural analyses of the 3D structures of native and mutant proteins confirmed the impact of these nsSNPs on the stability of the protein. Finally, the experimental results demonstrated that the structural stability of the mutant proteins was worse than that of the native protein. This study provides significant guidance for narrowing down the number of KRAS mutations to be screened as potential diagnostic biomarkers and to better understand the structural and functional mechanisms of the KRAS protein.

Computational Molecular Phenotypic Analysis of PTPN22 (W620R), IL6R (D358A), and TYK2 (P1104A) Gene Mutations of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder of bone joints caused by the complex interplay between several factors like body physiology, the environment with genetic background. The recent meta-analysis of GWAS has expanded the total number of RA-associated loci to more than 100, out of which approximately ∼97% (98 variants) loci are located in non-coding regions, and the other ∼3% (3 variants) are in three different non-HLA genes, i.e., TYK2 (Prp1104Ala), IL6R (Asp358Ala), and PTPN22 (Trp620Arg). However, whether these variants prompt changes in the protein phenotype with regards to its stability, structure, and interaction with other molecules, remains unknown. Thus, we selected the three clinically pathogenic variants described above, as positive controls and applied diverse computational methods to scrutinize if those mutations cause changes in the protein phenotype. Both wild type and mutant protein structures of PTPN22 (W620R), IL6R (D358A), and TYK2 (P1104A) were modeled and studied for structural deviations. Furthermore, we have also studied the secondary structure characteristics, solvent accessibility and stability, and the molecular interaction deformities caused by the amino acid substitutions. We observed that simple nucleotide predictions of SIFT, PolyPhen, CADD and FATHMM yields mixed findings in screening the RA-missense variants which showed a ≥P-value threshold of 5 × 10−8 in genome wide association studies. However, structure-based analysis confirms that mutant structures shows subtle but significant changes at their core regions, but their functional domains seems to lose wild type like functional interaction. Our findings suggest that the multidirectional computational analysis of clinically potential RA-mutations could act as a primary screening step before undertaking functional biology assays.

A possible founder mutation in FZD6 gene in a Turkish family with autosomal recessive nail dysplasia

BackgroundAutosomal recessive nail dysplasia is characterized by thick and hard nails with a very slow growth on the hands and feet. Mutations in FZD6 gene were found to be associated with autosomal recessive nail dysplasia in 2011. Presently, only seven mutations have been reported in FZD6 gene; five mutations are clustered in the C-terminus, one is at the seventh transmembrane domain, and another is at the very beginning of third extracellular loop.MethodsWhole exome sequencing (WES) was applied to the index case, her one affected sister and her healthy consanguineous parents. The mutation was verified via Sanger sequencing. Molecular dynamics simulations of the predicted structures of native and mutant proteins were compared to gain insight into the pathogenicity mechanism of the mutation.ResultsHere, we report a homozygous 8 bp deletion mutation, p.Gly559Aspfs*16; c.1676_1683delGAACCAGC, in FZD6 gene which causes a frameshift and creates a premature stop codon at position 16 of the new reading frame. Our molecular dynamics calculations predict that the pathogenicity of this frameshift mutation may be caused by the change in entropy of the protein with negative manner, disturbing the C-terminal domain structure, and hence interaction partners of FZD6.ConclusionWe identified a homozygous deletion mutation in FZD6 in a consanguineous Turkish family with nail dysplasia. We also provide a molecular mechanism about the effects of the deletion on the protein structure and its possible motions. This study provides a pathogenicity mechanism for this mutation in nail dysplasia for the first time.

Phenotypic and genetic analysis of two pedigrees affected with hereditary coagulation FXII deficiency

To carry out phenotypic and genotypic analysis for two Chinese pedigrees affected with coagulation factor XII (F XII) deficiency. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), and blood coagulation factor VIII, IX, XI, XII activity (FVIII:C, FIX:C, FXI:C, FXII:C) were determined with one stage clotting assay on a STAGO coagulation analyzer. FXII antigen was determined with an enzyme linked immunosorbent assay (ELISA). The 14 exons and their flanking sequences of the F12 gene were subjected to PCR amplification and Sanger sequencing. The conservation and structure of mutant protein were analyzed with MegAlign software and PYMOL software. The APTT of the probands was significantly prolonged, while their FXII:C and FXII:Ag were significantly reduced. Genetic analysis of the proband has revealed three novel mutations in the F12 gene, including g.5972G>A splice site mutation in intron 5, g.8810_8814delGTCTA in exon 14, and g.6259G>A (p.Pro182Leu) in exon 7. In addition, a previously known mutation IVS13-1G>A has been found. Four mutations have been identified in the two Chinese pedigrees, among which three were novel. Above mutations probably played a role in the defect of FXII in the two pedigrees.

Clinical features and identification of gene mutation in families with familial exudative vitreoretinopathy

Objective To identify mutations in NDP, FZD4, LRP5, TSPAN12 in Chinese families with familial exudative vitreoretinopathy (FEVR) and observe the clinical features. Methods Retrospective case series study. The 9 patients (18 eyes) and 5 normal members from 4 unrelated families were included in the study. The patients medical history and family history were collected in detail. All patients underwent best corrected visual acuity (BCVA), slit-lamp biomicroscopy, fundus colorized photography, fundus fluorescein angiography (FFA). Genomic DNA were collected from all the patients. Mutations were detected by directly sequencing to the whole coding region and exon-intron boundaries of NDP, FZD4, LRP5 and TSPAN12 gene. Polyphen and SIFT programs were used to predict the effects on the structure and functional properties of mutant protein. Results There were two affected individuals in the family 2 carried LRP5 gene mutation [c.1330C> T(p.R444C )] in exon 6 by sequence analysis. A score of 0.882 was acquired by Polyphen program analysis. And the missense change was predicted to be pathogenic by SIFT. Fundus changes of the proband showed angioplasia, tortuosity of peripheral vessels. And temporal dragging of the optic disc, peripheral avascular zone, neovascularization were found in FFA. Brush-like and straight of peripheral vessels were found in Ⅰ1. No variant was found in NDP, FZD4 and TSPAN12 gene. Conclusion Our study supports the gene mutation c.1330C> T (p.R444C) of LRP5 is pathogenesis of FEVR. Patients with the same mutation could have variable phenotypic characteristics. Key words: Retinal diseases/genetics; Retinal diseases/etiology; Genes; Mutation; Sequence analysis

Structural basis of 5' flap recognition and protein-protein interactions of human flap endonuclease 1.

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5′ flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein–protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5′ flap prior to its threading into the active site for cleavage. We also found that the β-pin region is rigidified into a short helix in R192F hFEN1–DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the β-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.

Expression, purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His6-tag system could be used to express ZNF191(243–368). The presence of the His6-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His6-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.

Analysis of DSPP gene mutation in a Chinese pedigree affected with hereditary dentinogenesis imperfecta

To analyze the clinical phenotype of a Chinese pedigree affected with hereditary dentinogenesis imperfecta and mutation of dentin sialophosphoprotein (DSPP) gene. Affected members underwent intraoral photography, dental film and panoramic radiography. Genomic DNA was extracted from peripheral venous blood samples. Coding regions of the DSPP gene were subjected to PCR amplification and Sanger sequencing. Functional effect of the mutation was predicted with SIFT and PolyPhen-2. The tertiary structure of wild type and mutant proteins were predicted by Swiss-Port. A heterozygous c.50C to T (p.P17L) mutation was identified in exon 2 of the DSPP gene in the proband and her father. The same mutation was not found among 200 unrelated healthy controls. The Pro-17 residues and its surrounding positions in DSPP are highly conserved across various species. The mutation was predicted to be damaging to the structure of DSPP protein. The c.50C to T (p.P17L) mutation of the DSPP gene probably underlies the disease in this pedigree. Above finding has expanded the spectrum of DSPP gene mutations and provided a basis for genetic counseling and prenatal diagnosis for this family.

Abstract CT033: Safety and early efficacy signals for COTI-2, an orally available small molecule targeting p53, in a phase I trial of recurrent gynecologic cancer

Abstract Background: Mutated p53 tumor suppressor protein is involved in &amp;gt;50% of all malignancies; however, only recently have candidate drugs designed to target p53 mutations entered clinical trials. COTI-2, an orally bioavailable "third-generation" thiosemicarbazone, was shown to restore the structure and function of mutant p53 proteins and induced growth inhibition in a number of p53 mutant cell lines. Thus, a three-part phase I dose escalation trial of COTI-2 was initiated with Part 1 reported herein. Methods: A multicenter, open label, 3+3 dose escalation trial enrolling patients with recurrent gynecological cancers with adequate performance status and bone marrow function. Patients could have unlimited prior therapies. Patients received COTI-2 5x/week orally over a 28-day cycle. Disease response was assessed by RECIST 1.1 every 8 weeks. Results: Twenty-four patients received study treatment: 19 ovarian, 3 cervical, and 2 endometrial cancers. Median age was 60 years (45-73 yrs) and patients had received a median of 5 prior chemotherapy regimens. After 4 dose cohorts (0.25, 0.5, 1.0, and 1.7 mg/kg), the Recommended phase II Dose (R2PD) for gynecologic cancer was 1.0 mg/kg. Dose-limiting toxicities (DLT) were encountered at 1.0 mg/kg (G3 abdominal pain, G3 sensory peripheral neuropathy, n=1) and 1.7 mg/kg, (G3 neuralgia n=1, G3 myalgia n=1). All DLTs resolved off treatment. Most common adverse events (&amp;gt; 10%) were nausea (67%), vomiting (67%), fatigue (54%), abdominal pain (46%), constipation (29%), anemia (29%), dyspnea (29%), anorexia (29%), urinary tract infection (21%), and hypokalemia (20%), myalgia (16%), diarrhea (16%), pyrexia (17%), peripheral neuropathy (17%), increased creatinine (13%), and weight loss (13%). Pharmacokinetic assessment showed oral administration of COTI-2 yielded a Tmax between 15-90 minutes and a half-life of 8-10 hours. Fifteen patients were evaluable for efficacy, having received at least one cycle of treatment. Of these, 13 had ovarian, 1 had cervical, and 1 had endometrial cancer. Eight patients had received one or more bevacizumab combinations, and two had received immune checkpoint inhibitors. Following COTI-2 treatment, 10 patients showed signs of possible activity: 1 patient had stable disease, 4 had stable target lesions, and 5 had stable non-target lesions. Twelve patients had comprehensive genomic profiling by next generation sequencing: 3 had wild-type p53 and 9 had either hotspot or non-hotspot mutations. Conclusions: COTI-2 was deemed generally safe and well-tolerated. Part 2 of the study is currently underway in HNSCC patients, with additional data expected in 2018, and Part 3 will follow with expansion to combination treatment of ovarian and HNSCC patients. Clinical trial information: NCT02433626. Citation Format: Shannon N. Westin, Wilberto Nieves-Neira, Christian Lynam, Kowthar Y. Salim, Alison D. Silva, Richard T. Ho, Gordon B. Mills, Robert L. Coleman, Filip Janku, Daniela Matei. Safety and early efficacy signals for COTI-2, an orally available small molecule targeting p53, in a phase I trial of recurrent gynecologic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT033.