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DYT-TOR1A is the most common inherited dystonia caused by a three nucleotide (GAG) deletion (dE) in the TOR1A gene. Death early after birth and cortical anomalies of the full knockout in rodents underscore its developmental importance. We therefore explored the timed effects of TOR1A-wt and TOR1A-dE during differentiation in a human neural in vitro model. We used lentiviral tet-ON expression of TOR1A-wt and -dE in induced neural stem cells derived from healthy donors. Overexpression was induced during proliferation of neural precursors, during differentiation and after differentiation into mature neurons. Overexpression of both wildtype and mutated protein had no effect on the viability and cell number of neural precursors as well as mature neurons when initiated before or after differentiation. However, if induced during differentiation, overexpression of TOR1A-wt and -dE led to a pronounced reduction of mature neurons in a dose dependent manner. Our data underscores the importance of physiological expression levels of TOR1A as crucial for proper neuronal differentiation. We did not find evidence for a specific impact of the mutated TOR1A on neuronal maturation. Electronic supplementary material The online version of this article (10.1007/s11033-020-05390-x) contains supplementary material, which is available to authorized users.

A Monte Carlo simulation based sequence design method is proposed to investigate the role of site-directed point mutations in protein misfolding. Site-directed point mutations are incorporated in the designed sequences of selected proteins. While most mutated sequences correctly fold to their native conformation, some of them stabilize in other nonnative conformations and thus misfold/unfold. The results suggest that a critical number of hydrophobic amino acid residues must be present in the core of the correctly folded proteins, whereas proteins misfold/unfold if this number of hydrophobic residues falls below the critical limit. A protein can accommodate only a particular number of hydrophobic residues at the surface, provided a large number of hydrophilic residues are present at the surface and critical hydrophobicity of the core is preserved. Some surface sites are observed to be equally sensitive toward site-directed point mutations as the core sites. Point mutations with highly polar and charged amino acids increases the misfold/unfold propensity of proteins. Substitution of natural amino acids at sites with different number of nonbonded contacts suggests that both amino acid identity and its respective site-specificity determine the stability of a protein. A clash-match method is developed to calculate the number of matching and clashing interactions in the mutated protein sequences. While misfolded/unfolded sequences have a higher number of clashing and a lower number of matching interactions, the correctly folded sequences have a lower number of clashing and a higher number of matching interactions. These results are valid for different SCOP classes of proteins.

Graphical abstract, Highlights • Study reveals specific dynamics of oncogenic K-Ras mutants G12C/V, G13D and Q61H. • P-loop mutations have pan-mutant and mutant-specific effects on local dynamics. • All active K-Ras P-loop mutations cause α3 helix to move away from Loop 7. • Only G12 mutations cause SII to move away from the α3 helix and the P-loop. • All studied mutations cause the α2 of SII region to get closer to C-terminal of α3., K-Ras is the most frequently mutated protein in human cancers. However, until very recently, its oncogenic mutants were viewed as undruggable. To develop inhibitors that directly target oncogenic K-Ras mutants, we need to understand both their mutant-specific and pan-mutant dynamics and conformations. Recently, we have investigated how the most frequently observed K-Ras mutation in cancer patients, G12D, changes its local dynamics and conformations (Vatansever et al., 2019). Here, we extend our analysis to study and compare the local effects of other frequently observed oncogenic mutations, G12C, G12V, G13D and Q61H. For this purpose, we have performed Molecular Dynamics (MD) simulations of each mutant when active (GTP-bound) and inactive (GDP-bound), analyzed their trajectories, and compared how each mutant changes local residue conformations, inter-protein distance distributions, local flexibility and residue pair correlated motions. Our results reveal that in the four active oncogenic mutants we have studied, the α2 helix moves closer to the C-terminal of the α3 helix. However, P-loop mutations cause α3 helix to move away from Loop7, and only G12 mutations change the local conformational state populations of the protein. Furthermore, the motions of coupled residues are mutant-specific: G12 mutations lead to new negative correlations between residue motions, while Q61H destroys them. Overall, our findings on the local conformational states and protein dynamics of oncogenic K-Ras mutants can provide insights for both mutant-selective and pan-mutant targeted inhibition efforts.

The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes. Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands. Fil: Campos, Ludmila Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina Fil: Garibotto, Francisco Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina Fil: Angelina, Emilio Luis. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas y Naturales y Agrimensura. Departamento de Química. Laboratorio de Estructura Molecular y Propiedades; Argentina Fil: Kos, Jiri. Univerzita Palackého v Olomouci; República Checa Fil: Tomašič, Tihomir. Univerza v Ljubljani; Eslovenia Fil: Zidar, Nace. Univerza v Ljubljani; Eslovenia Fil: Kikelj, Danijel. Univerza v Ljubljani; Eslovenia Fil: Gonec, Tomas. Veterinární a farmaceutická univerzita Brno; República Checa Fil: Marvanova, Pavlina. Veterinární a farmaceutická univerzita Brno; República Checa Fil: Mokry, Petr. Veterinární a farmaceutická univerzita Brno; República Checa Fil: Jampilek, Josef. Univerzita Palackého v Olomouci; República Checa. Univerzita Komenského v Bratislave; Eslovaquia Fil: Alvarez, Sergio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina Fil: Enriz, Ricardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina

Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of ABCB4 mutations and low biliary phospholipid concentration with symptomatic and recurring cholelithiasis. In the present study, we reported a case of a 63-year-old woman, who presented a biliary pain beginning at the age of 30, recurrent after cholecystectomy, along with “comet-tail shadows” revealed by ultrasonography thus, fulfilling the diagnosis of LPAC. This disease evolved into a cholangiocarcinoma. To understand the molecular basis of this phenotype, we performed the ABCB4 gene sequencing, followed by in silico analysis and Q-RT-PCR assay. The results displayed a homozygous missense sequence variation (c.140G > A, p.Arg47Gln), predicted as pathogenic according to MutPred. Accordingly, this gave rise to a decreased hepatic ABCB4 mRNA level and structural alterations of the mutated protein. Eventually, we reported, here, the first description of an ABCB4 missense mutation (p.Arg47Gln) at homozygous state in a Tunisian LPAC syndrome. An elucidation of its functional consequences was performed. Besides, this case suggests that the delayed diagnosis of LPAC syndrome and the lack of UDCA treatment may contribute in the development of complications, such as cholangiocarcinoma.

Parkinson’s disease (PD) is a neurodegenerative disorder resulting from the death of midbrain dopaminergic neurons and currently remains incurable despite enormous research efforts. The development of induced pluripotent stem cell (iPSC) technology offers the unique possibility to study disease mechanisms in any human tissue including patient-derived dopaminergic neurons. Of particular interest are the inherited monogenic forms of PD as they closely resemble the more common “idiopathic” PD and, through the mutated protein, provide a clear research target. This review provides an overview on the findings of iPSC-based genetic PD studies and explores them for unifying theories underlying PD disease mechanisms.

Background: P53 is the most commonly mutated protein in cancer with more than 40% of all cancer patients harboring a p53 mutation. Up to 85% of triple-negative breast cancers (TNBCs), aggressive breast cancers characterized by the absence of ER, PR, and Her2 receptors, have p53 mutations. P53 is a tumor suppressor that helps balance cellular growth and death after a genomic insult. Due to the rate of p53 mutation in cancer, the targeting of p53 in a clinical setting is an attractive option. However, currently there are no available p53-targeted drugs. We hypothesized that molecular pathways exist, that when inhibited, induce death or suppress growth of p53-mutant breast cancer cells, without affecting p53 wild-type breast cells. To investigate this hypothesis, we conducted a high-throughput drug screen to identify drugs that induce death or suppress growth of p53-mutant TNBCs. Methods: A library of 453 drugs with diverse and known mechanisms of action was used to perform high-throughput drug screening through collaboration with Texas A&M’s Center for Translational Cancer Research. An ATP luminescence assay was used to test eight TNBC cell lines with distinct p53 mutations in this screen. We then tested a subset of these drugs, identified to have an effect on p53-mutant TNBC, on the growth of five p53 wild-type breast cancer cell lines. Cell death assays to differentiate between growth suppression and death-inductive phenotypes are currently underway. Results: 145 drugs were identified that induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Of these drugs, 16 drugs were found to differentially suppress the growth of p53-mutant breast cancer cells, as compared to p53-wild type breast cancer cells. Further analysis of these drugs identified proteasome, apoptosis, and iron-dependent cell death pathways as potential p53-mutant specific targets in TNBCs. Conclusion: A screen of molecularly annotated drugs was performed in p53-mutant and p53-wild type breast cancer cells to identify drugs that preferentially kill or suppress the growth of p53-mutant breast cancers. These studies identified 145 drugs that were able to induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Testing these drugs in p53 wild-type breast cancer cell lines demonstrated that several of the identified drugs suppress growth or induce death preferentially in p53-mutant, as compared to p53 wild-type, breast cancers. These drugs affect specific cellular pathways such as proteasomal degradation, ferroptosis, and apoptosis pathways, which appear to be critical for the growth and survival of p53-mutant breast cancers. Further work will elucidate the interplay between p53-mutation and the identified cellular pathways. These studies will provide the basic science foundation for the development of new drugs for the treatment of p53-mutant breast cancer. This work was supported by a generous gift from the John Charles Cain Family. Citation Format: William Miska Tahaney, Reid Powell, Nghi Nguyen, Lakshmi Reddy Bollu, Jing Qian, Abhijit Mazumdar, Clifford Stephan, Peter JA Davies, Powel H Brown. Identification of drugs that induce the death or suppress the growth of p53-mutant breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-08.

International audience; A structural database of 11 families of chains differing by a single amino acid substitution has been built. Another structural dataset of 5 families with identical sequences has been used for comparison. The RMSD computed after a global superimposition of the mutated protein on each native one is smaller than the RMSD calculated among proteins of identical sequences. The effect of the perturbation is very local, and not necessarily the highest at the position of the mutation. A RMSD between mutated and native proteins is computed over a 3‐residue or a 7‐residue window at each position. To separate the effects of structural fluctuations due to point mutations from other sources, pair RMSD have been translated into P values which themselves are included in a score called P‐RANK. This score allows highlighting small backbone distortions by comparing these RMSD between mutated and native positions to the RMSD at the same positions in the absence of a mutation. It results from the P‐RANK that 38% of all mutations produce a significant effect on the displacement. When compared with a random distribution of RMSD at un‐mutated positions, we show that, even if the RMSD is greater when the mutation is in loops than in regular secondary structure, the relative effect is more important for regular secondary structures and for buried positions. We confirm the absence of correlation between RMSD and the predicted variation of free energy of folding but we found a small correlation between high RMSD and the error in the prediction of ΔΔG.

Compounds have been found that reduce levels of the harmful protein present in Huntington’s disease, without affecting the normal version. The compounds interact with the mutated protein and the cell’s protein-clearance machinery. Compounds that enable selective autophagy of mutant Huntingtin protein.

This paper describes the sequencing protocol and computational pipeline for the PGV-001 personalized vaccine trial. PGV-001 is a therapeutic peptide vaccine targeting neoantigens identified from patient tumor samples. Peptides are selected by a computational pipeline which identifies mutations from tumor/normal exome sequencing and ranks mutant sequences by a combination of predicted Class I MHC affinity and abundance estimated from tumor RNA. The PGV pipeline is modular and consists of many independently usable tools and software libraries. We draw attention to three particular tools which may be useful to other groups working on neoantigen vaccination. (1) Epidisco is a workflow which orchestrates the parallel execution of the PGV pipeline, including both common steps such as alignment as well as tools which have been developed specifically for the PGV-001 trial. (2) Vaxrank uses somatic variants and tumor RNA reads to select peptides for inclusion in a patient’s vaccine. (3) Isovar is a library used by Vaxrank to determine the mutated protein sequence associated with a genomic variant from supporting tumor RNA reads. We hope that the functionality of these tools may extend beyond the specifics of the PGV-001 trial and enable other research groups in their own neoantigen investigations.

Different approaches have been utilized or proposed for the treatment of lysosomal storage disorders (LSDs) including enzyme replacement and hematopoietic stem cell transplant therapies, both aiming to compensate for the enzymatic loss of the underlying mutated lysosomal enzymes. However, these approaches have their own limitations and therefore the vast majority of LSDs are either still untreatable or their treatments are inadequate. Missense mutations affecting enzyme stability, folding and cellular trafficking are common in LSDs resulting often in low protein half-life, premature degradation, aggregation and retention of the mutant proteins in the endoplasmic reticulum. Small molecular weight compounds such as pharmaceutical chaperones and proteostasis regulators have been in recent years shown to be promising approaches for overcoming some of these protein processing defects. These compounds are thought to enhance lysosomal enzyme activity by specific binding to the mutated enzyme or by manipulating components of the proteostasis pathways promoting protein stability, folding and trafficking and thus enhancing and restoring some of the enzymatic activity of the mutated protein in lysosomes. Multiple compounds have already been approved for clinical use to treat multiple LSDs like Migalastat in the treatment of Fabry disease and others are currently under research or in clinical trials such as Ambroxol hydrochloride and Pyrimethamine. In this review, we are presenting a general overview of LSDs, their molecular and cellular bases, and focusing on recent advances on targeting and manipulation proteostasis, including the use of pharmaceutical chaperones and proteostasis regulators, as therapeutic targets for some LSDs. In addition, we present the successes, limitations and future perspectives in this field.

The surface of the envelope spike of the human immunodeficiency virus (HIV) is covered with a dense array of glycans, which is sufficient to impede the host antibody response while maintaining a window for receptor recognition. The glycan density significantly exceeds that typically observed on self glycoproteins and is sufficiently high to disrupt the maturation process of glycans, from oligomannose- to complex-type glycosylation, that normally occurs during glycoprotein transit through the secretory system. It is notable that this generates a degree of homogeneity not seen in the highly mutated protein moiety. The conserved, close glycan packing and divergences from default glycan processing give a window for immune recognition. Encouragingly, in a subset of individuals, broadly neutralizing antibodies (bNAbs) have been isolated that recognize these features and are protective in passive-transfer models. Here, we review the recent advances in our understanding of the glycan shield of HIV and outline the strategies that are being pursued to elicit glycan-binding bNAbs by vaccination.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and has a mortality rate of 50% despite surgery. The major risk factors associated with HNSCC are smoking, alcohol, and human papillomavirus (HPV). p53, a transcriptional factor, is the most commonly mutated protein in HNSCC and plays an important and early role in tumorigenesis. The chapter highlights the story of p53 in the progression and management of HNSCC. In particular, we address p53’s mutational landscape and its resultant phenotypic outcomes. In addition, p53 as a prognostic biomarker and predictive biomarker for clinical outcome is addressed. Finally, we discuss p53 as a druggable target in HNSCC patients. This chapter aims to expand the understanding of the role of p53 in HNSCC in order to improve management of HNSCC patients by providing them with a personalized and customizable treatment plan.

Background Extensive use of NGS profiling in precision oncology is uncovering a staggering amount of novel alterations in the sequenced genes. Strikingly, the number of variants of uncertain significance (VUS) increases linearly with the addition of sequenced samples. While some of the VUS are inconsequential mutations many other VUS lead to activation of the proteins and are therefore involved in oncogenicity and may cause drugs resistance. These new activating mutations can also serve as novel drug targets. Methods We report here a method for functional annotation of these VUS as well as their sensitivity to targeted therapy. The system is based on rapid synthesis of the mutations and expression of the mutated protein together with fluorescently labeled reporters in a cell-based assay. The assay allows the accurate measurement of signaling pathway activation using fluorescence imaging coupled with high content image analysis. This process can also be performed with targeted agents to determine the sensitivity of the mutation. The output of the system is the level of activity of the mutant relative to a mutation with known activity in the same gene. Results Our results show that this method is able to accurately identify known driver mutations in 10 different oncogenes spanning 4 major cancer associated signaling pathways as well as over 100 different VUS in these genes. We also extend this analysis and show that the method correctly recapitulates differential sensitivity of EGFR mutations to 2nd and 3rd generation EGFR inhibitors and that different classes of BRAF mutations differ in response to specific inhibitors. Finally, we establish the importance of concurrent mutations in the response to EGFR inhibitors. Conclusions Taken together, this system provides crucial information for the development of new targeted agents and establishes an experimental tool both for patient stratification into clinical trials and patient treatment. Legal entity responsible for the study The authors. Funding NovellusDx. Disclosure Z. Barbash: Full / Part-time employment: NovellusDx. G. Tarcic: Full / Part-time employment: NovellusDx. D. Haham: Full / Part-time employment: NovellusDx. L. Hafzadi: Full / Part-time employment: NovellusDx. R. Zipor: Full / Part-time employment: NovellusDx. S. Barth: Full / Part-time employment: NovellusDx. A. Aizenman: Full / Part-time employment: NovellusDx.

Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein structure studies possible with a real-time PCR instrument address core topics in biochemistry and have valuable high-throughput applications in the fields of drug discovery and protein engineering. Protein analysis using real-time PCR instrumentation has been incorporated in an undergraduate laboratory project based on previously described projects. Students express, purify, and characterize a protein. Based on literature research and analysis using bioinformatics tools, they select a specific mutation to investigate. They then attempt to express, purify, and characterize their mutated protein. Thermal denaturation using a real-time PCR instrument is the primary tool used to compare the wild-type and mutated proteins. Alternative means for incorporation of protein analysis by real-time PCR instrumentation into laboratory experiences and additional modes of analysis are also described.

Over the past 20 years, the biological sciences have increasingly incorporated chemistry, physics, computer science, and mathematics to aid in the development and use of mathematical models. Such combined approaches have been used to address problems from protein structure–function relationships to the workings of complex biological systems. Computer simulations of molecular events can now be accomplished quickly and with standard computer technology. Also, simulation software is freely available for most computing platforms, and online support for the novice user is ample. We have therefore created a molecular dynamics laboratory module to enhance undergraduate student understanding of molecular events underlying organismal phenotype. This module builds on a previously described project in which students use site-directed mutagenesis to investigate functions of conserved sequence features in members of a eukaryotic protein kinase family. In this report, we detail the laboratory activities of a MD module that provide a complement to phenotypic outcomes by providing a hypothesis-driven and quantifiable measure of predicted structural changes caused by targeted mutations. We also present examples of analyses students may perform. These laboratory activities can be integrated with genetics or biochemistry experiments as described, but could also be used independently in any course that would benefit from a quantitative approach to protein structure–function relationships. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(6):402–408, 2013

Although the mutated protein causing Huntington's disease (HD) is expressed throughout the body, the major pathology of HD is localized to the striatum of the brain. We previously reported that the striatal-enriched protein Rhes binds the mutated huntingtin protein and enhances its cytotoxicity. We now demonstrate that Rhes-deleted mice are dramatically protected from neurotoxicity and motor dysfunction in a striatal-specific model of HD elicited by 3-nitropropionic acid. This finding suggests that Rhes may, in part, determine the striatal selectivity of HD.

The proteins produced in the body control and mediate the metabolic processes and help in its routine functioning. Any kind of impairment in protein production, such as production of mutated protein, or misfolded protein, leads to disruption of the pathway controlled by that protein. This may manifest in the form of the disease. However, these diseases can be treated, by supplying the protein from outside or exogenously. The supply of active exogenous protein requires its production on large scale to fulfill the growing demand. The process is complex, requiring higher protein expression, purification, and processing. Each product needs unique settings or standardizations for large-scale production and purification. As only large-scale production can fulfill the growing demand, thus it needs to be cost-effective. The tools of genetic engineering are utilized to produce the proteins of human origin in bacteria, fungi, insect, or mammalian host. Usage of recombinant DNA technology for large-scale production of proteins requires ample amount of time, labor, and resources, but it also offers many opportunities for economic growth. After reading this chapter, readers would be able to understand the basics about production of recombinant proteins in various hosts along with the advantages and limitations of each host system and properties and production of some of the important pharmaceutical compounds and growth factors.

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.

Objectives This review groups the newest results of molecular analyses of DSPP gene for patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and tries to link the phenotypes with specific mutations in the DSPP gene. Data The review includes biochemical data introducing a specificity of DSPP protein which justifies it as a critical factor for dentine mineralization and maturation. The majority of the review analyzes mutations in the DSPP gene which result in phenotypes of dentinogenesis imperfecta types II or/and III or dentine dysplasia. Sources An electronic search was conducted in the databases of Pub Med and supplemented by manual study of relevant references. Study selection 52 out of 108 references were finally selected for the review based on the novelty and/or originality of data. Conclusion Hereditary dentine disorders dentinogenesis imperfecta type II/III and dentine dysplasia are currently proposed to be one disease with distinct clinical manifestations reflecting various mutations in the same DSPP gene. For years both disorders were linked exclusively to mutations in the DSP code but a growing number of papers describe mutations which manifest a similar phenotype but are localized in the strongly repetitive sequence of the 3′ terminus of the DSPP which codes DPP protein. Our search suggests that the localization of mutation in the sequence of the DSPP gene might result in a different phenotype due to the diverse cellular fate of the mutated protein. Thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is still required.

Diabet. Med. 28, 1476–1486 (2011) Abstract Sir Harold Himsworth first observed and articulated the phenomenon of insulin resistance in the late 1930s. Although a long delay followed before his observations were acknowledged and enshrined in formal diagnostic classifications of diabetes mellitus, insulin resistance-related pathology in the early 21st century poses one of the major global healthcare challenges for contemporary physicians. Whilst insulin resistance is closely related to obesity and decreased physical fitness, despite intensive investigation it has proved extremely challenging to discriminate key events in its causation from epiphenomena, many related to compensation for the primary defect. Thus, a complete account of the molecular pathogenesis of insulin resistance-related diseases remains elusive. One approach circumventing such problems is the study of patients with single gene defects causing severe insulin resistance. In such patients the primary defect is known, and thus lessons may be learned about human physiology from detailed physiological study allied to knowledge of the function of the mutated protein. This review discusses developments in understanding of monogenic severe insulin resistance since discovery of the first insulin receptor mutations in 1988 and reviews the physiological lessons learnt, including the critical role of adipose tissue in human metabolic health and the meaning and importance of ‘partial’ insulin resistance for major human disease.

Neurodegeneration Parkin is a commonly mutated protein ubiquitin ligase implicated in Parkinson's disease. Understanding how Parkin functions is important to understanding its pathogenic role. In a Perspective, Arkinson and Walden discuss recent structural insights into the activities of Parkin.

The KRAS protein is known to play a key role in various oncogenic pathways. KRAS mutations are found in 20-30 % of patients with non-small cell lung cancer (NSCLC). The majority of mutations are found at KRAS codons 12 and 13, and they appear to be more frequent in smokers and adenocarcinoma. The mutated protein is in its active state despite absence of stimulation, which leads to the constitutive activation of downstream pathways responsible for cellular disorder. The identification of new biomarkers to predict the evolution of cancer pathogenesis (predictive factor) and the sensibility to treatments (predictive factor) is one of the major objectives in oncology research. KRAS mutations are a potential candidate biomarker and numerous studies have tried to confirm its place as a prognostic/predictive biomarker in NSCLC. The results are contradictory and most studies are retrospective. The first results of prospective studies are currently reported, in particular with the use of antibodies against EGFR. The exact place of KRAS in medical thoracic oncology remains to be determined and further studies are needed. To date, KRAS mutations are not a biomarker to be used routinely.

The torsion dystonias encompass a broad collection of etiologic subtypes, often divided into primary and secondary classes. Over the last two decades an increasing number of genetic causes have been identified, including an important genetic cause for early-onset primary torsion dystonia (PTD): a GAG deletion in exon 5 of DYT1 , a gene that encodes torsinA. Although the exact function of torsinA remains elusive, evidence suggests aberrant localization and interaction of mutated protein; this may result in an abnormal response to stress or interference with cytoskeletal events and the development of neuronal brain pathways. Identification of DYT1 has also permitted studies of both “manifesting” and “non-manifesting” DYT1 mutation carriers. These investigations have expanded our understanding of clinical expression to include psychiatric symptoms and also have enabled imaging studies of endophenotypes. Similarly, there has been progress in our understanding of the genetic underpinnings of the “dystonia-plus” syndromes: dopa-responsive dystonia (DRD), myoclonus–dystonia (M-D), and rapid-onset dystonia–parkinsonism (RDP). These advances provide a widened platform for future research.

Defective bone mineralization has serious clinical manifestations, including deformities and fractures, but the regulation of this extracellular process is not fully understood. We have developed a mathematical model consisting of ordinary differential equations that describe collagen maturation, production and degradation of inhibitors, and mineral nucleation and growth. We examined the roles of individual processes in generating normal and abnormal mineralization patterns characterized using two outcome measures: mineralization lag time and degree of mineralization. Model parameters describing the formation of hydroxyapatite mineral on the nucleating centers most potently affected the degree of mineralization, while the parameters describing inhibitor homeostasis most effectively changed the mineralization lag time. Of interest, a parameter describing the rate of matrix maturation emerged as being capable of counter-intuitively increasing both the mineralization lag time and the degree of mineralization. We validated the accuracy of model predictions using known diseases of bone mineralization such as osteogenesis imperfecta and X-linked hypophosphatemia. The model successfully describes the highly non-linear mineralization dynamics, which includes an initial lag phase when osteoid is present but no mineralization is evident, then fast primary mineralization, followed by secondary mineralization characterized by a continuous slow increase in bone mineral content. The developed model can potentially predict the function for a mutated protein based on the histology of pathologic bone samples from mineralization disorders of unknown etiology.

B-Raf is the most frequently mutated protein kinase in human cancer. Thus, inhibition of B-Raf kinase activity could benefit cancer patients. However, the clinical benefit of RAF inhibitor therapie...

The p8 protein is involved in the cellular stress response of many tissues. Because p8 is overexpressed in many cancers, we investigated whether its expression was required for tumour development. Mouse embryo fibroblasts (MEFs) from p8+/+ and p8–/– animals were transformed with the pBabe-rasV12/E1A retroviral vector, which expresses both the rasV12 mutated protein and the E1A oncogene. As expected, transformed p8+/+ MEFs could form colonies in soft agar. However, transformed p8–/– MEFs could not. In addition, subcutaneous or intraperitoneal injections of transformed p8+/+ MEFs always led to tumour formation in nude mice, but, again, no tumour was observed with transformed p8–/– MEFs. However, restoring p8 expression in transformed p8–/– MEFs before injection led to tumour formation. In the tumours, p8 expression was induced during tumour development. It was concluded that p8 expression in transformed MEFs is necessary for tumour formation, suggesting that the stress-response mechanisms governed by p8 are required for tumour establishment.

Introduction: Cancer of the breast is one of the commonest carcinomas in women, both in western world and in India. The high frequency of breast cancer in women has prompted an intensive study of possible modifiable risk factors (clinical parameters, morphological typing, and biological markers) for assessment of prognosis, prevention strategies, and treatment modalities. p53 is one of the most significant prognostic markers for breast carcinomas. Aims and Objectives: This study was undertaken to ascertain the prevalence of p53 mutated protein in breast carcinomas by immunohistochemistry and correlating the results with clinical and histological parameters and also determine its influence on axillary node metastasis. Materials and Methods: Clinico-pathological evaluation of 50 cases of infiltrating ductal carcinoma of the breast, with reference to p53 mutated protein expression by means of immunohistochemistry was undertaken. Results: The percentage positivity of p53 immunohistochemistry of this study was 22% which is similar to the reported statistics of various other studies. The immunohistochemistry results also showed an equivocal p53 status in both the positive and negative cases of lymph node metastases. The carcinomas with p53 positivity demonstrated aggressive characteristics, including larger size, higher grade, in comparison with p53 negative cases. The probability of getting positive p53 status was higher for those with severe lymphocytic reaction of tumours, for patients less than 35 years of age and for those with tumour on the left side. This study clearly indicates that p53 over expression in breast cancers are mostly aggressive tumours and they confer poor prognosis and likelihood of a poor response to endocrine therapy and chemotherapy.

We report the case of a young adolescent with ataxia telangiectasia (AT) and life-threatening haemorrhage from the bladder due to a combination of bladder wall telangiectasis, immunosuppressive therapy and an infection with polyomavirus JC. BK and JC are both members of the polyomavirus family. BK virus is a known cause of haemorrhagic cystitis in bone-marrow and nephropathy in kidney transplant patients, whereas JC virus is mainly associated with progressive multifocal leukoencephalopathy and only rarely found in haemorrhagic cystitis. Although opportunistic infections are uncommon in AT and virus replication was described as being down-regulated in ATM (AT mutated protein)-deficient cells, clinicians should be aware that severe haematuria in a patient with AT and undergoing immunosuppressive therapy is suggestive for polyomavirus JC-induced haemorrhagic cystitis.

With the development of deep DNA sequencing techniques, the cost for detecting mutations in the human genome falls significantly. Numerous non-synonymous single nucleotide polymorphisms (nsSNPs) have been identified and many of them are associated with human disease. One of the long-standing challenges is to understand how nsSNPs change protein structure and further affect their function. While it is impractical to solve all the mutated protein structures experimentally, it is quite feasible to model the mutated structures in silico. Toward this goal, we are building a publicly available structure database (SNP2Structure) to facilitate our research endeavors. Compared with the existing web portals with a similar aim, ours has three major advantages. First, we corrected the existing sequence mapping discrepancies presented in others. Although the percentage of erroneously mapped structures is small, it is critical to correct such errors. Second, our portal offers comparison of two structures simultaneously. Third, the mutated structures are available to download locally for further investigations. We believe SNP2Structure will be a valuable tool to the research community to understand the functional impact of disease-causing nsSNPs.

In this paper we present the data of lymphocyte radiosensitivity testing used for characterization of radiosensitive cellular phenotype and diagnostics of ataxia-telangiectasia disease. We point out the advantage of lymphocyte micronucleus test (CBMN) over other cellular tests for assessment of radiosensitivity: the first advantage of CBMN is that primary patient cells are used (less than 1 ml), the second one is that the results of testing are obtained within 3 days and there is no need for establishing a patient-derived cell line, which requires additional time and application of more expensive methods. The third advantage of CBMN method is that it gives information about proliferative ability of cells, which can recognize dysfunctional ataxia-telangiectasia mutated protein. The results are fast and accurate in diagnostics of ataxia-telagiectasia diseases.

Despite its well-defined causality, we still don’t know precisely how the polymorphic expansion of the CAG repeat in the first exon of the huntingtin gene results in the pleiotropic, devastating, and ultimately fatal pathophysiology of Huntington’s disease. One long-standing theory posits that the mutated protein adversely affects the cellular machinery that maintains energetic homeostasis and in so doing results in a variety of untoward downstream consequences. A large number of studies have yielded mixed results, with some supporting the ‘energetic hypothesis’ while others have not found convincing evidence for this mechanism. Two large interventional clinical trials of compounds involved in energy production (2 CARE for CoQ10 and CREST for creatine) both failed to meet their efficacy endpoints and provided no additional insights for the selection or design of subsequent energetic drug candidates. Unlike well-defined mitochondriopathies, the purported energetic changes in HD are likely to be subtle, chronic, and progressive. As such, experimental medicine studies in humans that use sensitive and specific measurements need to be conducted to build a highly detailed molecular understanding of any energetics-related deficits in HD-affected individuals. These studies will be critical to refine our mechanistic hypothesis and rationally select targets for pharmacological intervention, design shorter informative trials, and increase the confidence that future drug candidates will benefit patients. Existing data and plans for prospective studies consistent with this strategy will be presented.

Vascular anomalies are localized abnormalities that occur during vascular development. Several causative genes have been identified not only for inherited but also for some sporadic forms, and the molecular pathways involved are becoming understood. This gives us the opportunity to generate animals carrying the causative genetic defects, which we hope model the phenotype seen in human patients. These models would enable us not only to test known antiangiogenic drugs, but also to develop novel approaches for treatment, directly targeting the mutated protein or molecules implicated in the pathophysiological signaling pathways.

Deafness is the most common form of sensory impairment in humans. Several mutations have been reported in the myosin VI proteins that are responsible for autosomal dominant and recessive deafness. One such missense mutation is that which changes glutamate (E) to valine (V) at amino acid 216 (E216V). The mutation resides in the myosin VI head domain; however, detailed insights regarding the impact of this mutation on structural stability are not known. To characterize the potential impact of this mutation on protein stability and its association with deafness, we performed computational sequence and structural analysis of normal and mutated myosin VI. Overall, protein stability declined when the mutation was introduced, as predicted by I-mutant and Cologne University Protein Stability Analysis Tool (CUPSAT). Similarly, a decline in hydrophobicity around position 216 was observed.Phosphorylation of myosin VI was also observed to be influenced by the E216V mutation. Furthermore, a three-dimensional structure of myosin VI was studied using Swiss-PdbViewer to identify structural variations in the protein that may occur as a result of the E216V mutation. Distortion in the hydrogen bonding between Val216 and Ser449 in the mutated protein structure was observed, exposing the Ser449 hydroxyl group and reducing overall protein stability. Key words: Myosin VI, genetics, deafness, hereditary mutations, structure analysis.

Models of the 3D structure of full-length human CFTR described during the last years have provided new insights into the molecular basis of CFTR functioning. They have in particular shed light on the interdomain interfaces which are critical for signal transmission and can thus be considered for in silico screening of drugs (correctors and modulators) capable of filling the cavities displayed at these interfaces in the wild type or mutated protein. However, the modeling methodology does largely influence the characteristics of those interdomain interfaces. Here, relying on all the available experimental data on ABC transporters (full length and isolated domains), we show that there is a considerable structural plasticity in these regions which allows to adapt the partner domains. The careful consideration of such a structural plasticity in our modeling strategy leads us to propose at present four models of human CFTR, two (based on the Sav1866 and MsbA structures) in a similar open channel conformation and two (based on the MsbA and P-gp structures) in notably different closed channel conformations. We interestingly show that the global features of the transmission interfaces [between the membrane-spanning domains (MSDs) and the nucleotide binding domains (NBDs)] are remarkably conserved in these models between the open and closed forms. Further investigation of the details of the MSDs/NBDs interface also suggests that subtle differences may participate in the general mechanisms by which the signal is transmitted between MSDs and NBDs. Supported by the association Vaincre La Mucoviscidose, Paris, France.

ATM (Ataxia-telangiectasia mutated protein kinase) mediates DNA double-stranded break (DSB) signaling. ATM deficiency results in the autosomal recessive disorder known as ataxia-telangiectasia (A-T...

International audience; The catalytic domain of the acetylcholinesterases is composed of a single polypeptide chain, the folding of which determines two subdomains. We have linked these two subdomains by mutating two residues, I327 and D375, to cysteines, to form a disulfide bridge. As a consequence, the hydrodynamic radius of the protein was reduced, suggesting that there is some flexibility in the subdomain connection. In addition to the smaller size, the mutated protein is more stable than the wild-type protein. Therefore, the flexibility between the two domains is a weak point in terms of protein stability. As expected from the location of the disulfide bond at the rim of the active site, the kinetic studies show that it affects interactions with peripheral ligands and the entrance of some of the bulkier substrates, like o-nitrophenyl acetate. In addition, the mutations affect the catalytic step for o-nitrophenyl acetate and phosphorylation by organophosphates, suggesting that this movement between the two subdomains is connected with the cooperativity between the peripheral and catalytic sites.

Identification of SOD1 as the mutated protein in a significant subset of familial amyotrophic lateral sclerosis (FALS) cases has led to the generation of transgenic rodent models of autosomal dominant SOD1 FALS. Mice carrying 23 copies of the human SOD1(G93A) transgene are considered the standard model for FALS and ALS therapeutic studies. To date, there have been at least 50 publications describing therapeutic agents that extend the lifespan of this mouse. However, no therapeutic agent besides riluzole has shown corresponding clinical efficacy. We used computer modeling and statistical analysis of 5429 SOD1(G93A) mice from our efficacy studies to quantify the impact of several critical confounding biological variables that must be appreciated and should be controlled for when designing and interpreting efficacy studies. Having identified the most critical of these biological variables, we subsequently instituted parameters for optimal study design in the SOD1(G93A) mouse model. We retested several compounds reported in major animal studies (minocycline, creatine, celecoxib, sodium phenylbutyrate, ceftriaxone, WHI-P131, thalidomide, and riluzole) using this optimal study design and found no survival benefit in the SOD1(G93A) mouse for any compounds (including riluzole) administered by their previously reported routes and doses. The presence of these uncontrolled confounding variables in the screening system, and the failure of these several drugs to demonstrate efficacy in adequately designed and powered repeat studies, leads us to conclude that the majority of published effects are most likely measurements of noise in the distribution of survival means as opposed to actual drug effect. We recommend a minimum study design for this mouse model to best address and manage this inherent noise and to facilitate more significant and reproducible results among all laboratories employing the SOD1(G93A) mouse.

Wild-type iso-1-cytochrome c from Saccharomyces cerevisiae containing naturally occurring cysteine at position 102 and mutated protein S47C (derived from the protein in which C102 had been replaced by threonine) were labeled with cysteine-specific methanethiosulfonate spin label. Continuous wave (CW) electron paramagnetic resonance (EPR) was used to examine the effect of temperature on the behavior of the spin label in the oxidized and reduced forms of wild-type cytochrome c and in the oxidized form of the mutated protein. The computer simulations revealed that the CW EPR spectrum for each form of cytochrome c consists of at least two components [a fast (F) and a slow (S) component], which differ in the values of the rotational correlation times $$\tau _{{\rm R}_\parallel } $$ (longitudinal rotational correlation time) and $$\tau _{{\rm R}_ \bot } $$ (transverse rotational correlation time) and that the relative contributions of the F and S components of the spectra change with temperature. In addition, the values of the rotational correlation times ( $$\tau _{{\rm R}_\parallel } $$ and $$\tau _{{\rm R}_ \bot } $$ ) for the F component appear to change much more dramatically with the temperature than the respective values for the S component. A large difference between the behavior of the oxidized and reduced wild-type spin-labeled cytochromes c indicates that the temperature-induced unfolding of the protein in the region around C102 progresses more rapidly when cytochrome c is in the oxidized form.

We have shown that the thymopoietin-like octapeptides derived from DNA-binding domain of p53 protein and of its mutated forms differ in their immunomodulatory properties. A strong increase of immunostimulative activity was observed for GMNRSPIL (mutated protein) in comparison with GMNRRPIL (wild-type of p53 protein) peptide. Here the elongated sequences of respective protein fragments were synthesized and investigated by plaque forming cells and delayed type hypersensitivity tests. The change of immunomodulatory activity toward immunosuppression was observed: NSSC(Acm)MGGMNRRPILTIITLE (I, wild-type) was inactive in both tests, and the C(Acm)MGGMNRSPILTIITLE (II) and YMC(Acm)NSSC(Acm)MGGMNRSPILTIITLE (III) (mutated p53 protein fragments) peptides produced immunosuppression in plaque forming cells as well as in delayed type hypersensitivity tests.

A McGill University–led team has shown that CFTR corrector compounds target only one of two sequential steps required for proper folding of the mutated protein. The findings suggest there is a clear rationale for developing combinations of correctors to treat patients with cystic fibrosis.