Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization

2014 ◽  
Vol 459 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Jesper R. van Dijk ◽  
Yasuo Yamazaki ◽  
Ruth H. Palmer
Keyword(s):  
Point Mutations ◽  
Ubiquitin Ligases ◽  
Mutant Proteins ◽  
Pa Domain

In the present study, we have identified a role for the PA domain of the RNF13/RNF167 ubiquitin ligases, showing that it is both sufficient and required for endosomal localization. Furthermore, although PA domain point-mutations identified from tumours are ligase active, PA mutant proteins are mislocalized within the cell.

Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src

1990 ◽  
Vol 10 (4) ◽  
pp. 1307-1318
Author(s):  
H Hirai ◽  
H E Varmus
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Kinase Domain ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Positive Effects ◽  
Src Gene ◽  
Carboxy Terminal

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.

scholarly journals The Primary Causes of Muscle Dysfunction Associated with the Point Mutations in Tpm3.12; Conformational Analysis of Mutant Proteins as a Tool for Classification of Myopathies

2018 ◽  
Vol 19 (12) ◽  
pp. 3975 ◽  
Author(s):  
Yurii Borovikov ◽  
Olga Karpicheva ◽  
Armen Simonyan ◽  
Stanislava Avrova ◽  
Elena Rogozovets ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Fiber Type ◽  
Point Mutations ◽  
Nemaline Myopathy ◽  
Muscle Dysfunction ◽  
Thin Filaments ◽  
Mutant Proteins ◽  
Strong Binding ◽  
Genes Encoding

Point mutations in genes encoding isoforms of skeletal muscle tropomyosin may cause nemaline myopathy, cap myopathy (Cap), congenital fiber-type disproportion (CFTD), and distal arthrogryposis. The molecular mechanisms of muscle dysfunction in these diseases remain unclear. We studied the effect of the E173A, R90P, E150A, and A155T myopathy-causing substitutions in γ-tropomyosin (Tpm3.12) on the position of tropomyosin in thin filaments, and the conformational state of actin monomers and myosin heads at different stages of the ATPase cycle using polarized fluorescence microscopy. The E173A, R90P, and E150A mutations produced abnormally large displacement of tropomyosin to the inner domains of actin and an increase in the number of myosin heads in strong-binding state at low and high Ca2+, which is characteristic of CFTD. On the contrary, the A155T mutation caused a decrease in the amount of such heads at high Ca2+ which is typical for mutations associated with Cap. An increase in the number of the myosin heads in strong-binding state at low Ca2+ was observed for all mutations associated with high Ca2+-sensitivity. Comparison between the typical conformational changes in mutant proteins associated with different myopathies observed with α-, β-, and γ-tropomyosins demonstrated the possibility of using such changes as tests for identifying the diseases.

scholarly journals Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael Bucher ◽  
Stephan Niebling ◽  
Yuhao Han ◽  
Dmitry Molodenskiy ◽  
Fatemeh Hassani Nia ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Hippocampal Neurons ◽  
Tertiary Structure ◽  
Point Mutations ◽  
Autism Spectrum ◽  
Missense Mutations ◽  
Glutamatergic Synapses ◽  
Mutant Proteins ◽  
Synaptic Targeting ◽  
Induced Changes

Members of the SH3- and ankyrin-rich repeat (SHANK) protein family are considered as master scaffolds of the post-synaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons.

scholarly journals S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) ReduceIn VitroInhibition by Fluconazole

2012 ◽  
Vol 56 (4) ◽  
pp. 2099-2107 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Jonathan G. L. Mullins ◽  
Claire M. Hull ◽  
Josie E. Parker ◽  
David C. Lamb ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Point Mutations ◽  
Triazole Ring ◽  
Wild Type ◽  
Protein Activity ◽  
Content Type ◽  
Mutant Proteins ◽  
Type Protein ◽  
Wild Type Protein ◽  
Binding Spectra

ABSTRACTThe effects of S279F and S279Y point mutations inCandida albicansCYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (Ks, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (Ks, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higherKdvalues observed.

scholarly journals Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition.

1997 ◽  
Vol 17 (5) ◽  
pp. 2436-2447 ◽  
Author(s):  
E Alani ◽  
T Sokolsky ◽  
B Studamire ◽  
J J Miret ◽  
R S Lahue
Keyword(s):  
Base Pair ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Atp Binding ◽  
Mutant Proteins ◽  
Mismatch Recognition

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches. In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes. A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstream of mismatch recognition. Helix-turn-helix domain mutant proteins displayed two different properties. One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches. A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein. Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition. We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.

scholarly journals The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

Acta Naturae ◽  
2015 ◽  
Vol 7 (1) ◽  
pp. 87-97 ◽  
Author(s):  
O. V. Orlova ◽  
V. L. Drutsa ◽  
P. V. Spirin ◽  
A. V. Ivanov ◽  
V. S. Prasolov ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Point Mutations ◽  
Envelope Proteins ◽  
Protein Glycosylation ◽  
Virus Envelope ◽  
Mutant Proteins ◽  
Virus Like Particles ◽  
E2 Glycoprotein ◽  
Glycosylation Sites

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

scholarly journals S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity

2021 ◽  
Vol 15 (1) ◽  
pp. e0009007
Author(s):  
Fábio Mambelli ◽  
Bruno P. O. Santos ◽  
Suellen B. Morais ◽  
Enrico G. T. Gimenez ◽  
Duana C. dos S. Astoni ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Neutrophil Elastase ◽  
Point Mutations ◽  
Point Of View ◽  
Mutant Proteins ◽  
Inflammatory Parameters ◽  
C Terminus ◽  
Kunitz Domain

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD’s inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

Compound Heterozygous Protein C Deficiency Caused by Two Mutations, Arg-178 to Gin and Cys-331 to Arg, Leading to Impaired Secretion of Mutant Protein C

1994 ◽  
Vol 72 (06) ◽  
pp. 814-818 ◽  
Author(s):  
Yuichi Sugahara ◽  
Osamu Miura ◽  
Shinsaku Hirosawa ◽  
Nobuo Aoki
Keyword(s):  
Disulfide Bond ◽  
Protein C ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Expression Vectors ◽  
Compound Heterozygous ◽  
Protein C Deficiency ◽  
Peptide Cleavage ◽  
Mutant Proteins ◽  
Heterozygous Protein C Deficiency

SummaryThe protein C gene in a patient apparently homozygous for protein C deficiency was analyzed. Two different point mutations, each located in a different allele, were detected to reveal that the patient is a compound heterozygote. Mutation of Arg-178 (CGG) to Gin (CAG) [mutation I] was detected in exon VII, in the vicinity of activation peptide cleavage site by thrombin. Mutation of Cys-331 (TGC) to Arg (CGC) [mutation II] was found in exon IX, at one of the sites involved in disulfide bond formation in the catalytic domain of the heavy chain. The alteration of Cys-331 to Arg disables the formation of the disulfide bond and would alter the protein conformation. Transient expression assays using COS-7 cells transfected with protein C expression vectors containing each one of these two mutations suggested that each of the two mutations would lead to the protein C deficiency by an impairment of secretion of the respective mutant proteins.

scholarly journals PLP1 Mutations in Patients with Multiple Sclerosis: Identification of a New Mutation and Potential Pathogenicity of the Mutations

2018 ◽  
Vol 7 (10) ◽  
pp. 342 ◽  
Author(s):  
Nancy Cloake ◽  
Jun Yan ◽  
Atefeh Aminian ◽  
Michael Pender ◽  
Judith Greer
Keyword(s):  
Multiple Sclerosis ◽  
Clinical Symptoms ◽  
Point Mutations ◽  
Human Leukocyte ◽  
In Silico Analysis ◽  
Proteolipid Protein ◽  
Wild Type ◽  
Exon 2 ◽  
Mutant Proteins ◽  
The Unfolded Protein Response

PLP1 is located on the X-chromosome and encodes myelin proteolipid protein (PLP), the most abundant protein in central nervous system myelin. Generally, point mutations in PLP1 result in X-linked dysmyelinating disorders, such as Pelizaeus-Merzbacher disease (PMD) or spastic paraplegia type 2 (SPG2). However, several case studies have identified patients with missense point mutations in PLP1 and clinical symptoms and signs compatible with a diagnosis of multiple sclerosis (MS). To investigate if PLP1 mutations occur relatively frequently in MS, we sequenced the coding regions of PLP1 in 22 female MS patients who had developed disease after the age of 40 and in 42 healthy women, and identified a missense mutation in exon 2 of PLP1 resulting in a Leu30Val mutation in the protein in one of the MS patients. mCherry-tagged plasmids containing wild type or mutant PLP1 sequences of PLP, including two known PMD/SPG2-related mutations as positive controls, were constructed and transfected into Cos-7 cells. In comparison with cells transfected with wild type PLP1, all mutations caused significant accumulation of PLP in the endoplasmic reticulum of the cells and induction of the unfolded protein response—a mechanism that leads to apoptosis of cells expressing mutant proteins. Additionally, in silico analysis of the binding of peptides containing the Leu30Val mutation to the human leukocyte antigen (HLA) molecules carried by the patient harboring this mutation suggested that the mutation could produce several novel immunogenic epitopes in this patient. These results support the idea that mutations in myelin-related genes could contribute to the development of MS in a small proportion of patients.

Evidence for an interaction between adducin and Na+-K+-ATPase: relation to genetic hypertension

1999 ◽  
Vol 277 (4) ◽  
pp. H1338-H1349 ◽  
Author(s):  
Mara Ferrandi ◽  
Sergio Salardi ◽  
Grazia Tripodi ◽  
Paolo Barassi ◽  
Rodolfo Rivera ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Specific Interaction ◽  
Point Mutations ◽  
Kidney Medulla ◽  
Mutant Proteins ◽  
Cytoskeleton Organization ◽  
Pig Kidney ◽  
Genetic Hypertension ◽  
Actin Cytoskeleton Organization

Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na+-K+-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na+-K+-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na+-K+-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na+-K+-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E2 (K) → E1 (Na) or E2(K) ⋅ ATP → E1Na ⋅ ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human α- and β-adducins stimulate Na+-K+-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na+-K+-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na+-K+-ATPase, which does not contain adducin, added adducin stimulates the Na+-K+-ATPase activity of microsomes only about one-half as much as that of purified Na+-K+-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na+-K+-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

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