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Prediction of disease-related mutations affecting protein localization
Kirsti Laurila, Mauno Vihinen
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-122
Abstract: Numerous methods have been developed to predict protein subcellular localization with quite high accuracy. We applied bioinformatics methods to investigate the effects of known disease-related mutations on protein targeting and localization by analyzing over 22,000 missense mutations in more than 1,500 proteins with two complementary prediction approaches. Several hundred putative localization affecting mutations were identified and investigated statistically.Although alterations to localization signals are rare, these effects should be taken into account when analyzing the consequences of disease-related mutations.Eukaryotic cells contain numerous compartments, such as cytoplasm, mitochondria, Golgi apparatus, and peroxisomes, all of which contain different protein constituents and have different functions. Proteins are typically directed to these compartments by short peptide sequences that act as targeting signals. For example, secretory, chloroplast and mitochondrial targeting peptides are located at the N terminus, whereas signals for other compartments can be within the amino acid sequence. Terminal signal peptides are typically cleaved during the protein translocation process.Protein function depends on numerous factors. One important but often neglected property is its subcellular localization. Translocation to the proper compartment allows a protein to form the necessary interactions with its partners and take part in biological networks. For example, signalling and metabolic pathways are dependent on the location of the constituent proteins. Failure to be transported to the correct intracellular compartment can have detrimental effects, which appear in different ways. Either the reaction performed or information carried by the protein does not reach the proper site, causing either inactivation of central reactions or misregulation of, eg, signalling cascades, or the mislocalized protein is active, but has harmful effects by acting in the wrong place.Subcel
The BRCA1 Breast Cancer Suppressor: Regulation of Transport, Dynamics, and Function at Multiple Subcellular Locations  [PDF]
Beric R. Henderson
Scientifica , 2012, DOI: 10.6064/2012/796808
Abstract: Inherited mutations in the BRCA1 gene predispose to a higher risk of breast/ovarian cancer. The BRCA1 tumor suppressor is a 1863 amino acid protein with multiple protein interaction domains that facilitate its roles in regulating DNA repair and maintenance, cell cycle progression, transcription, and cell survival/apoptosis. BRCA1 was first identified as a nuclear phosphoprotein, but has since been shown to contain different transport sequences including nuclear export and nuclear localization signals that enable it to shuttle between specific sites within the nucleus and cytoplasm, including DNA repair foci, centrosomes, and mitochondria. BRCA1 nuclear transport and ubiquitin E3 ligase enzymatic activity are tightly regulated by the BRCA1 dimeric binding partner BARD1 and further modulated by cancer mutations and diverse signaling pathways. This paper will focus on the transport, dynamics, and multiple intracellular destinations of BRCA1 with emphasis on how regulation of these events has impact on, and determines, a broad range of important cellular functions. 1. Introduction The BRCA1 protein is classified as a tumor suppressor [1]; in healthy cells it functions to maintain proper genomic repair and cell division, but inherited mutations in the BRCA1 gene encode altered forms of the protein that contribute to development of breast and ovarian cancer [2–4]. Misregulation and reduced expression of BRCA1 also contribute to sporadic forms of breast cancer [5]. The primary tumor suppressing role of BRCA1 relates to the maintenance of genomic integrity through regulation of DNA replication, repair, and transcription, in addition to various cell cycle checkpoints that ensure survival of healthy cells [6]. BRCA1 gene mutations disrupt these processes and result in chromosome instability and defective checkpoints that accelerate cellular transformation [6–8]. BRCA1 is a multifunctional protein that binds dozens of other proteins, the most important of which is BARD1 [9–11] (see Figure 1(a)). BARD1 forms a stable heterodimer with BRCA1, stimulating its nuclear localization and ubiquitin E3 ligase activity. While the BARD1 gene, also regarded as a tumor suppressor, is susceptible to germ-line and somatic mutations, these occur at a much lower frequency in a subset of breast/ovarian cancers [12–14]. Figure 1: BRCA1 domain structure and subcellular transport pathways. (a) Protein domain structure of BRCA1 showing the location of nuclear localization signals (NLSs), nuclear export signals (NESs), and binding sites for BARD1 and gamma-tubulin. The RING and BRCT
Penetrance of eye defects in mice heterozygous for mutation of Gli3 is enhanced by heterozygous mutation of Pax6
Paulette A Zaki, J Martin Collinson, Junko Toraiwa, T Ian Simpson, David J Price, Jane C Quinn
BMC Developmental Biology , 2006, DOI: 10.1186/1471-213x-6-46
Abstract: We showed that Gli3 is expressed in the developing eye but that Gli3+/- mice have only very subtle eye defects. We then generated mice compound heterozygous for mutations in both Gli3 and Pax6, which encodes another developmentally important transcription factor known to be crucial for eye development. Pax6+/-; Gli3+/- eyes were compared to the eyes of wild-type, Pax6+/- or Gli3+/- siblings. They exhibited a range of abnormalities of the retina, iris, lens and cornea that was more extensive than in single Gli3+/- or Pax6+/- mutants or than would be predicted by addition of their phenotypes.These findings indicate that heterozygous mutations of Gli3 can impact on eye development. The importance of a normal Gli3 gene dosage becomes greater in the absence of a normal Pax6 gene dosage, suggesting that the two genes co-operate during eye morphogenesis.The zinc-finger transcription factor Gli3 is required for normal limb, brain and eye development. In humans, a number of different mutations to the GLI3 allele can cause Greig cephalopolysyndactyly (GCPS) or Pallister-Hall Syndrome (PHS) [1,2]. Clinical features of GCPS include polydactyly, syndactyly, ocular hypertelorism and macrocephaly; those of PHS include polydactyly, imperforate anus, renal abnormalities, hypothalamic hamartoma and pituitary dysplasia. Mutations of the Gli3 gene in mice cause the extra-toes (Xt) phenotype [3]. Johnson [4] first reported a comprehensive analysis of the developmental anatomy of Xt mice. Homozygotes die by birth and the most prominent defects are found in the distal limbs, in rostral portions of the head including the forebrain and eyes (see below), along the ventral midline of the thorax and along the midline of the visceral ectoderm. Heterozygotes are described as developing relatively normally in all respects bar the formation of an extra digit, or digit-like appendage, on either the fore- or hindlimbs.The vertebrate transcription factors Gli1, Gl2 and Gli3 are homologues of the Dros
GLI3 Repressor Controls Nephron Number via Regulation of Wnt11 and Ret in Ureteric Tip Cells  [PDF]
Jason E. Cain, Epshita Islam, Fiona Haxho, Lin Chen, Darren Bridgewater, Erica Nieuwenhuis, Chi-Chung Hui, Norman D. Rosenblum
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0007313
Abstract: Truncating GLI3 mutations in Pallister-Hall Syndrome with renal malformation suggests a requirement for Hedgehog signaling during renal development. HH-dependent signaling increases levels of GLI transcriptional activators and decreases processing of GLI3 to a shorter transcriptional repressor. Previously, we showed that Shh-deficiency interrupts early inductive events during renal development in a manner dependent on GLI3 repressor. Here we identify a novel function for GLI3 repressor in controlling nephron number. During renal morphogenesis, HH signaling activity, assayed by expression of Ptc1-lacZ, is localized to ureteric cells of the medulla, but is undetectable in the cortex. Targeted inactivation of Smo, the HH effector, in the ureteric cell lineage causes no detectable abnormality in renal morphogenesis. The functional significance of absent HH signaling activity in cortical ureteric cells was determined by targeted deletion of Ptc1, the SMO inhibitor, in the ureteric cell lineage. Ptc1?/?UB mice demonstrate ectopic Ptc1-lacZ expression in ureteric branch tips and renal hypoplasia characterized by reduced kidney size and a paucity of mature and intermediate nephrogenic structures. Ureteric tip cells are remarkable for abnormal morphology and impaired expression of Ret and Wnt11, markers of tip cell differentiation. A finding of renal hypoplasia in Gli3?/? mice suggests a pathogenic role for reduced GLI3 repressor in the Ptc1?/?UB mice. Indeed, constitutive expression of GLI3 repressor via the Gli3Δ699 allele in Ptc1?/?UB mice restores the normal pattern of HH signaling, and expression of Ret and Wnt11 and rescued the renal phenotype. Thus, GLI3 repressor controls nephron number by regulating ureteric tip cell expression of Wnt11 and Ret.
Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development
Amir A Abbasi, Zissis Paparidis, Sajid Malik, Fiona Bangs, Ansgar Schmidt, Sabine Koch, Javier Lopez-Rios, Karl-Heinz Grzeschik
BMC Developmental Biology , 2010, DOI: 10.1186/1471-213x-10-44
Abstract: Here, we demonstrate in chicken and mouse transgenic embryos that human GLI3-intronic conserved non-coding sequence elements (CNEs) autonomously control individual aspects of Gli3 expression. Their combined action shows many aspects of a Gli3-specific pattern of transcriptional activity. In the mouse limb bud, different CNEs enhance Gli3-specific expression in evolutionary ancient stylopod and zeugopod versus modern skeletal structures of the autopod. Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos. Three of these elements govern central nervous system specific gene expression during mouse embryogenesis, each targeting a subset of endogenous Gli3 transcription sites. Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly. During evolution, ancient broad-range regulatory elements within Gli3 attained higher specificity, critical for patterning of more specialized structures, by abolishing the potential for redundant expression control.These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.Zinc-finger proteins of the GLI family, GLI1, GLI2, and GLI3 act as transcriptional mediators integrating various upstream patterning signals in a context dependent combinatorial and cooperative fashion to direct a multitude of developmental programs. GLI2 and GLI3 can serve both as transcriptional activators or repressors, whereas GLI1, whose expression is transcriptionally regulated by GLI2 and GLI3, appears to play a secondary role, e.g. in potentiating response to the secreted protein sonic hedgehog (SHH) [1].Mutations in the human GLI3 gene cause a variety of dominant developmental syndromes subsumed as "GLI3 mo
Functional diversification of duplicate genes through subcellular adaptation of encoded proteins
Ana C Marques, Nicolas Vinckenbosch, David Brawand, Henrik Kaessmann
Genome Biology , 2008, DOI: 10.1186/gb-2008-9-3-r54
Abstract: We show that for 24-37% of duplicate gene pairs derived from the S. cerevisiae whole-genome duplication event, the two members of the pair encode proteins that localize to distinct subcellular compartments. The propensity of yeast duplicate genes to evolve new localization patterns depends to a large extent on the biological function of their progenitor genes. Proteins involved in processes with a wider subcellular distribution (for example, catabolism) frequently evolved new protein localization patterns after duplication, whereas duplicate proteins limited to a smaller number of organelles (for example, highly expressed biosynthesis/housekeeping proteins with a slow rate of evolution) rarely relocate within the cell. Paralogous proteins evolved divergent localization patterns by partitioning of ancestral localizations ('sublocalization'), but probably more frequently by relocalization to new compartments ('neolocalization'). We show that such subcellular reprogramming may occur through selectively driven substitutions in protein targeting sequences. Notably, our data also reveal that relocated proteins functionally adapted to their new subcellular environments and evolved new functional roles through changes of their physico-chemical properties, expression levels, and interaction partners.We conclude that protein subcellular adaptation represents a common mechanism for the functional diversification of duplicate genes.Gene duplication is an important evolutionary mechanism, providing genomes with the genetic raw material for the emergence of genes with new or altered functions [1]. Several evolutionary fates of the two duplicate gene copies are possible and have been described. For instance, one of the two copies may be redundant and accumulate deleterious mutations that eventually render it a non-functional pseudogene [1]. Alternatively, both copies might be functionally preserved by natural selection if an increase in gene dosage of the ancestral gene is benefic
Mutations in Global Regulators Lead to Metabolic Selection during Adaptation to Complex Environments  [PDF]
Gerda Saxer ,Michael D. Krepps,Eric D. Merkley,Charles Ansong,Brooke L. Deatherage Kaiser,Marie-Thérèse Valovska,Nikola Ristic,Ping T. Yeh,Vittal P. Prakash,Owen P. Leiser,Luay Nakhleh,Henry S. Gibbons,Helen W. Kreuzer,Yousif Shamoo
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004872
Abstract: Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes if many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased genetic and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator arcA and the stress response regulator rpoS. We identified 46 unique single-nucleotide variants of arcA and 18 mutations in rpoS, nine of which resulted in stop codons or large deletions, suggesting that subtle modulations of ArcA function and knockouts of rpoS are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order metabolic selection that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting E. coli populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism, and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators arcA and rpoS can provide a “one-step” mechanism of adaptation to a novel environment, which highlights the importance of global resource management as a powerful strategy to adaptation.
Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies  [PDF]
Benedikt Frieg?,Boris G?rg?,Nadine Homeyer?,Verena Keitel?,Dieter H?ussinger?,Holger Gohlke
PLOS Computational Biology , 2016, DOI: 10.1371/journal.pcbi.1004693
Abstract: Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.
Mutations in Planar Cell Polarity Gene SCRIB Are Associated with Spina Bifida  [PDF]
Yunping Lei, Huiping Zhu, Cody Duhon, Wei Yang, M. Elizabeth Ross, Gary M. Shaw, Richard H. Finnell
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069262
Abstract: Neural tube defects (NTDs) (OMIM #182940) including anencephaly, spina bifida and craniorachischisis, are severe congenital malformations that affect 0.5–1 in 1,000 live births in the United States, with varying prevalence around the world. Mutations in planar cell polarity (PCP) genes are believed to cause a variety of NTDs in both mice and humans. SCRIB is a PCP-associated gene. Mice that are homozygous for the Scrib p.I285K and circletail (Crc) mutations, present with the most severe form of NTDs, namely craniorachischisis. A recent study reported that mutations in SCRIB were associated with craniorachischisis in humans, but whether SCRIB mutations contribute to increased spina bifida risk is still unknown. We sequenced the SCRIB gene in 192 infants with spina bifida and 190 healthy controls. Among the spina bifida patients, we identified five novel missense mutations that were predicted-to-be-deleterious by the PolyPhen software. Of these five mutations, three of them (p.P1043L, p.P1332L, p.L1520R) significantly affected the subcellular localization of SCRIB. In addition, we demonstrated that the craniorachischisis mouse line-90 mutation I285K, also affected SCRIB subcellular localization. In contrast, only one novel missense mutation (p.A1257T) was detected in control samples, and it was predicted to be benign. This study demonstrated that rare deleterious mutations of SCRIB may contribute to the multifactorial risk for human spina bifida.
The Transcriptional Repressor Domain of Gli3 Is Intrinsically Disordered  [PDF]
Robert Tsanev, Kalju Vanatalu, Jüri Jarvet, Risto Tanner, Kristi Laur, Piret Tiigim?gi, Birthe B. Kragelund, Torben ?sterlund, Priit Kogerman
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076972
Abstract: The transcription factor Gli3 is acting mainly as a transcriptional repressor in the Sonic hedgehog signal transduction pathway. Gli3 contains a repressor domain in its N-terminus from residue G106 to E236. In this study we have characterized the intracellular structure of the Gli3 repressor domain using a combined bioinformatics and experimental approach. According to our findings the Gli3 repressor domain while being intrinsically disordered contains predicted anchor sites for partner interactions. The obvious interaction partners to test were Ski and DNA; however, with both of these the structure of Gli3 repressor domain remained disordered. To locate residues important for the repressor function we mutated several residues within the Gli3 repressor domain. Two of these, H141A and H157N, targeting predicted helical regions, significantly decreased transcriptional repression and thus identify important functional parts of the domain.
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