KinMutBase, a database of human disease-causing protein kinase mutations

KinMutBase (http://www.uta.fi/imt/bioinfo/KinMutBase/) is a registry of mutations in human protein kinases related to disorders. Kinases are essential cellular signaling molecules, in which mutations can lead to diseases, including immunodeficiencies, cancers and endocrine disorders. The first release of KinMutBase contained information for protein tyrosine kinases. The current release includes also serine/threonine protein kinases, as well as an update of the tyrosine kinases. There are 251 entries altogether, representing 337 families and 621 patients. Mutations appear both in conserved hallmark residues of the kinases as well as in non-homologous sites. The KinMutBase WWW pages provide plenty of information, namely mutation statistics and display, clickable sequences with mutations and changes to restriction enzyme patterns.     

Functional analysis of disease-causing mutations in human galactokinase.

Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia.     

Spectrum of disease-causing mutations in protein secondary structures

Background  

Most genetic disorders are linked to missense mutations as even minor changes in the size or properties of an amino acid can alter or prevent the function of the protein. Further, the effect of a mutation is also dependent on the sequence and structure context of the alteration.     

Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing

Lubricin, a protein product of the gene PRG4, is a secreted mucin-like proteoglycan that is a major lubricant in articulating joints. Mutations in PRG4 cause the autosomal recessive, human disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome. We developed rabbit polyclonal antibodies against human lubricin to determine the consequence of disease-causing mutations at the protein level and to study the protein's normal post-translational processing. Antiserum generated against an epitope in the amino-terminal portion of lubricin detected protein in wild-type synovial fluid and in conditioned media from wild-type cultured synoviocytes. However, the antiserum did not detect lubricin in synovial fluid or cultured synoviocytes from several patients with frameshift or nonsense mutations in PRG4. Antiserum generated against an epitope in the protein's carboxyl-terminal, hemopexin-like domain identified a post-translational cleavage event in wild-type lubricin, mediated by a subtilisin-like proprotein convertase (SPC). Interestingly, in contrast to wild-type lubricin, one disease-causing mutation that removes the last 8 amino acids of the protein, including a conserved cysteine residue, was not cleaved within the hemopexin-like domain when expressed in COS-7 cells. This suggests that formation of an intrachain disulfide bond is required for SPC-mediated cleavage and that SPC-mediated cleavage is essential to protein function.     

MKKS is a centrosome-shuttling protein degraded by disease-causing mutations via CHIP-mediated ubiquitination

McKusick–Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet–Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin–proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.     

Desmosomal protein structure and function and the impact of disease-causing mutations

In this graphical review we focus on the structural characteristics of desmosomal proteins, their interactions with each other and with the intermediate filament cytoskeleton. The wealth of structural information that is now available allows predictions to be made about the pathogenic effect of disease-causing mutations. We have selected representative examples of missense mutations that are buried, semi-buried or surface exposed, and demonstrate how such variants could affect the structural fold of desmosomal proteins that are expressed in the heart. We explain how such alterations could compromise desmosomal adhesion, resulting in life threatening diseases including arrhythmogenic right ventricular cardiomyopathy.     

Functional analysis of disease-causing mutations in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase (GALE, EC 5.1.3.2) catalyses the interconversion of UDP-glucose and UDP-galactose. Point mutations in this enzyme are associated with the genetic disease, type III galactosemia, which exists in two forms - a milder, or peripheral, form and a more severe, or generalized, form. Recombinant wild-type GALE, and nine disease-causing mutations, have all been expressed in, and purified from, Escherichia coli in soluble, active forms. Two of the mutations (N34S and G319E) display essentially wild-type kinetics. The remainder (G90E, V94M, D103G, L183P, K257R, L313M and R335H) are all impaired in turnover number (k cat) and specificity constant (k cat/Km), with G90E and V94M (which is associated with the generalized form of galactosemia) being the most affected. None of the mutations results in a greater than threefold change in the Michaelis constant (Km). Protein-protein crosslinking suggests that none of the mutants are impaired in homodimer formation. The L183P mutation suffers from severe proteolytic degradation during expression and purification. N34S, G90E and D103G all show increased susceptibility to digestion in limited proteolysis experiments. Therefore, it is suggested that reduced catalytic efficiency and increased proteolytic susceptibility of GALE are causative factors in type III galactosemia. Furthermore, there is an approximate correlation between the severity of these defects in the protein structure and function, and the symptoms observed in patients.     

Detecting disease-causing mutations in the human genome by haplotype matching

Comparisons between haplotypes from affected patients and the human reference genome are frequently used to identify candidates for disease-causing mutations, even though these alignments are expected to reveal a high level of background neutral polymorphism. This limits the scope of genetic studies to relatively small genomic intervals, because current methods for distinguishing potential causal mutations from neutral variation are inefficient. Here we describe a new strategy for detecting mutations that is based on comparing affected haplotypes with closely matched control sequences from healthy individuals, rather than with the human reference genome. We use theory, simulation, and a real data set to show that this approach is expected to reduce the number of sequence variants that must be subjected to follow-up analysis by at least a factor of 20 when closely matched control sequences are selected from a reference panel with as few as 100 control genomes. We also define a reference data resource that would allow efficient application of this strategy to large critical intervals across the genome.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

Twenty years of Alzheimer's disease-causing mutations

In this study, we review our finding of APP mutations in Alzheimer's disease in 1990-1991 with the benefit of 20 years' perspective. We discuss the historical context in which we made the finding, its immediate and continuing effects on research activity and our hopes for successful clinical testing of the hypothesis. We also briefly discuss the effects finding APP mutations has had on our own careers and those of our colleagues from 1991.     

Functional analysis of the neurofibromatosis type 2 protein by means of disease-causing point mutations

Despite intense study of the neurofibromatosis type 2 (NF2) tumor-suppressor protein merlin, the biological properties and tumor-suppressor functions of merlin are still largely unknown. In this study, we examined the molecular activities of NF2-causing mutant merlin proteins in transfected mammalian cells, to elucidate the merlin properties that are critical for tumor-suppressor function. Most important, we found that 80% of the merlin mutants studied significantly altered cell adhesion, causing cells to detach from the substratum. This finding implies a function for merlin in regulating cell-matrix attachment, and changes in cell adhesion caused by mutant protein expression may be an initial step in the pathogenesis of NF2. In addition, five different mutations in merlin caused a significant increase in detergent solubility of merlin compared to wild type, indicating a decreased ability to interact with the cytoskeleton. Although not correlated to the cell-adhesion phenotype, four missense mutations decreased the binding of merlin to the ERM-interacting protein EBP-50, implicating this interaction in merlin inhibition of cell growth. Last, we found that some NF2 point mutations in merlin most closely resembled gain-of-function alleles in their cellular phenotype, which suggests that mutant NF2 alleles may not always act in a loss-of-function manner, as had been assumed, but may include a spectrum of allelic types with different phenotypic effects on the function of the protein. In aggregate, these cellular phenotypes provide a useful assay for identifying the functional domains and molecular partners necessary for merlin tumor-suppressor activity.     

Characterization of a protein kinase from human seminal plasma.

Localization of creatine kinase in isolated cardiac cells nuclei has been studied. Carefully purified cardiac nuclei preparations contain creatine kinase electrophoretically similar to the mitochondrial creatine kinase isoenzyme. Histochemical electron microscopic investigations have shown that nuclear creatine kinase is localized inside nuclei closely to chromatin.     

Tyrosine kinase mutations in human cancer

A subset of tyrosine kinases are activated by mutations which contribute to the malignant transformation, growth, and metastasis of human cancers. Mutations change the expression, conformation and/or stability of tyrosine kinases, often leading to constitutive activation of the signaling pathways the kinases regulate. Given that tyrosine kinases are key members of signaling cascades, mutations have multiple effects on various cellular proteins. This review will focus on four kinases (EGFR, c-Met, c-Kit, and PI3-kinase) known to be mutated in human cancer. It will discuss the effects that these mutations have on the biology of tumors, and how our understanding of the structure and function of kinases and their mutations is currently being used to design targeted treatments.     

Alteration of CFTR transmembrane span integration by disease-causing mutations

Many missense mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) result in its misfolding, endoplasmic reticulum (ER) accumulation, and, thus, cystic fibrosis. A number of these mutations are located in the predicted CFTR transmembrane (TM) spans and have been projected to alter span integration. However, the boundaries of the spans have not been precisely defined experimentally. In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. The mutations either destabilize the integrated conformation or alter the TM1 ER integration profile. G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. By contrast, temperature-dependent misfolding owing to the G91R mutation depends on the introduction of the basic side chain rather than the loss of the glycine. This work demonstrates that CF-causing mutations predicted to have similar effects on CFTR structure actually result in disparate molecular perturbations that underlie ER accumulation and the pathology of CF.     

The structural context of disease-causing mutations in gap junctions

Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.     

Expression of TTK, a novel human protein kinase, is associated with cell proliferation.

We have isolated the full-length sequence for a unique human kinase, designated TTK. TTK was initially identified by screening of a T cell expression library with anti-phosphotyrosine antibodies. The kinases most closely related to TTK are the SPK1 serine, threonine and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase which was also identified through screening of an expression library with anti-phosphotyrosine antibodies. However, the relationships are distant with less than 25% identity. Nevertheless, TTK is highly conserved throughout phylogeny with hybridizing sequences being detected in mammals, fish, and yeast. TTK mRNA is present at relatively high levels in testis and thymus, tissues which contain a large number of proliferating cells, but is not detected in most other benign tissues. Freshly isolated cells from most malignant tumors assessed expressed TTK mRNA. As well, all rapidly proliferating cell lines tested expressed TTK mRNA. Escherichia coli expressing the complete kinase domain of TTK contain markedly elevated levels of phosphoserine and phosphothreonine as well as slightly increased levels of phosphotyrosine. Taken together, these findings suggest that expression of TTK, a previously unidentified member of the family of kinases which can phosphorylate serine, threonine, and tyrosine hydroxyamino acids, is associated with cell proliferation.