A shift in the salt bridge interaction of residues D620 and E621 mediates the constitutive activation of Jak2-H538Q/K539L

Jak2 mutations in the exon 14 and exon 12 regions that cause constitutive activation have been associated with myeloproliferative neoplasms. We have previously shown that a pi stacking interaction between F617 and F595 is important for the constitutive activation of Jak2-V617F (Gnanasambandan et al., Biochemistry 49:9972-9984, 2010). Here, using a combination of molecular dynamics (MD) simulations and in vitro mutagenesis, we studied the molecular mechanism for the constitutive activation of the Jak2 exon 12 mutation, H538Q/K539L. The activation levels of Jak2-H538Q/K539L were found to be similar to that of Jak2-V617F, and Jak2-H538Q/K539L/V617F. Data from MD simulations indicated a shift in the salt bridge interactions of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L. When compared to Jak2-WT, K539A mutation resulted in increased activation, while K539D or K539E mutations diminished Jak2 activation by 50 %. In the context of Jak2-H538Q/K539L, R541A mutation reduced its activation by 50 %, while R541D and R541E mutations returned its activation levels to that of Jak2-WT. Collectively, these results indicate that a shift in the salt bridge interaction of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L is critical for constitutive activation of this Jak2 exon 12 mutant.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Novel and recurrent mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia

We have identified 16 different mutations of the low-density lipoprotein receptor (LDLR) gene in 25 unrelated Korean patients with heterozygous familial hypercholesterolemia (FH), including five novel mutations, C83Y, 661del17, 1705insCTAG, C675X, and 941-1G>A. The 1705insCTAG mutation in which the four 3 cent -terminal nucleotides of exon 11 are duplicated was found to prevent splicing of exon 11 and would therefore generate a truncated polypeptide. The in-frame 36-bp deletion (1591del36) in exon 11, which had been reported only in one Korean FH patient, was also found. We showed that this change affects transport of the LDL receptor from the endoplasmic reticulum to the cell surface. In addition, we found 8 mutations (-136C>T, E119K, E207K, E207X, F382L, R574Q, 1846-1G>A, and P664L) that had been described in other ethnic groups but not in Koreans, and 2 mutations (R94H and D200N) that had been described in Koreans as well as other ethnic groups. 5 mutations (1591del36, E119K, E207X, E207K, and P664L) were found more than once in the Korean FH samples. Identification of the novel and recurring LDLR mutations in Korean FH patients should facilitate prenatal and early diagnosis in families at high risk of FH.     

The spectrum of mutations in the low-density lipoprotein receptor gene in the Russian population

The spectrum of mutations in the low-density lipoprotein (LDL) receptor gene was studied in a sample of hypercholesterolemia patients of Caucasoid origin from the population of Russia. The examined patients were 45 to 49 years old and had the highest level of total serum cholesterol in this age group. Seven previously nondescribed mutations have been revealed in exon 9 (R410G; M412V) and in exon 12 (Y/Y576; N/N591; L605V; L605R; A612G). Twelve previously described mutations have been identified in exons 2 (C/C27), 5 (C261F; E240X), 6 (E288K), 8 (A391T), 9 (E418G; L432R; D433E), 11 (G/G549; E558K; L/L568), and 12 (G592E). Only one of these mutations was previously described in Russia in a clinical sample of patients with familial hypercholesterolemia. The spectrum of LDL receptor gene mutations in the population sample of patients with hypercholesterolemia significantly differs from the mutation spectrum in patients with familial hypercholesterolemia (clinical samples). Sequencing of the LDL receptor gene is a highly efficient method for identifying the markers of hypercholesterolemia predisposition in a population.     

Fabry disease: thirty-five mutations in the alpha-galactosidase A gene in patients with classic and variant phenotypes.

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene located at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes and for precise carrier detection, the alpha-Gal A lesions in 42 unrelated Fabry hemizygotes were determined. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and their family members. The seven alpha-galactosidase A exons and flanking intronic sequences were PCR amplified and the nucleotide sequence was determined by solid-phase direct sequencing. RESULTS: Two patients with the mild cardiac phenotype had missense mutations, I9IT and F113L, respectively. In 38 classically affected patients, 33 new mutations were identified including 20 missense (MIT, A31V, H46R, Y86C, L89P, D92Y, C94Y, A97V, R100T, Y134S, G138R, A143T, S148R, G163V, D170V, C202Y, Y216D, N263S, W287C, and N298S), two nonsense (Q386X, W399X), one splice site mutation (IVS4 + 2T-->C), and eight small exonic insertions or deletions (304del1, 613del9, 777del1, 1057del2, 1074del2, 1077del1, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition, two unique complex rearrangements consisting of contiguous small insertions and deletions were found in exons 1 and 2 causing L45R/H46S and L120X, respectively. CONCLUSIONS: These studies further define the heterogeneity of mutations causing Fabry disease, permit precise carrier identification and prenatal diagnosis in these families, and facilitate the identification of candidates for enzyme replacement therapy.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

Increased alkali stability in Trichoderma reesei endo-1, 4-beta-xylanase II by site directed mutagenesis

A number of engineered Trichoderma reesei endo-beta-1,4-xylanase (Xyn II) mutants were created and activity tests were performed for increased stability. The stability of the earlier characterized mutant Y5 (T2C, T28C, K58R, +191D) was further increased by the mutations creating the constructs P9 (N97R+F93W+H144K), P12 (H144C+N92C), P15 (F180Q+H144C+N92C) and P21 (H22K+F180Q+H144C+N92C). The resistance towards thermal inactivation at alkaline pH was increased in all of the mutants. Residual activity T(50%) was increased 4-5 degrees C for P9 at pH 9. The performance of the P9 mutant in sulphate pulp bleaching was also tested and was shown to increase brightness markedly compared to the reference. The bleaching results showed the industrial potential of the obtained mutant.     

Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions

Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the α2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase α2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the α and β subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na⁺ and increase the K⁺ affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K⁺ and Na⁺ affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).     

Functional consequences of PRODH missense mutations

PRODH maps to 22q11 in the region deleted in the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) and encodes proline oxidase (POX), a mitochondrial inner-membrane enzyme that catalyzes the first step in the proline degradation pathway. At least 16 PRODH missense mutations have been identified in studies of type I hyperprolinemia (HPI) and schizophrenia, 10 of which are present at polymorphic frequencies. The functional consequences of these missense mutations have been inferred by evolutionary conservation, but none have been tested directly. Here, we report the effects of these mutations on POX activity. We find that four alleles (R185Q, L289M, A455S, and A472T) result in mild (<30%), six (Q19P, A167V, R185W, D426N, V427M, and R431H) in moderate (30%-70%), and five (P406L, L441P, R453C, T466M, and Q521E) in severe (>70%) reduction in POX activity, whereas one (Q521R) increases POX activity. The POX encoded by one severe allele (T466M) shows in vitro responsiveness to high cofactor (flavin adenine dinucleotide) concentrations. Although there is limited information on plasma proline levels in individuals of known PRODH genotype, extant data suggest that severe hyperprolinemia (>800 microM) occurs in individuals with large deletions and/or PRODH missense mutations with the most-severe effect on function (L441P and R453C), whereas modest hyperprolinemia (300-500 microM) is associated with PRODH alleles with a moderate reduction in activity. Interestingly, three of the four alleles associated with or found in schizophrenia (V427M, L441P, and R453C) resulted in severe reduction of POX activity and hyperprolinemia. These observations plus the high degree of polymorphism at the PRODH locus are consistent with the hypothesis that reduction in POX function is a risk factor for schizophrenia.     

SCH28080, a K+-competitive inhibitor of the gastric H,K-ATPase,binds near the M5-6 luminal loop,preventing K+ access to the ion binding domain

Inhibition of the gastric H,K-ATPase by the imidazo[1,2-alpha]pyridine, SCH28080, is strictly competitive with respect to K+ or its surrogate, NH4+. The inhibitory kinetics [V(max), K(m,app)(NH4+), K(i)(SCH28080), and competitive, mixed, or noncompetitive] of mutants can define the inhibitor binding domain and the route to the ion binding region within M4-6. While mutations Y799F, Y802F, I803L, S806N, V807I (M5), L811V (M5-6), Y928H (M8), and Q905N (M7-8) had no effect on inhibitor kinetics, mutations P798C, Y802L, P810A, P810G, C813A or -S, I814V or -F, F818C, T823V (M5, M5-6, and M6), E914Q, F917Y, G918E, T929L, and F932L (M7-8 and M8) reduced the affinity for SCH28080 up to 10-fold without affecting the nature of the kinetics. In contrast, the L809F substitution in the loop between M5 and M6 resulted in an approximately 100-fold decrease in inhibitor affinity, and substitutions L809V, I816L, Y925F, and M937V (M5-6, M6, and M8) reduced the inhibitor affinity by 10-fold, all resulting in noncompetitive kinetics. The mutants L811F, Y922I, and I940A also reduced the inhibitor affinity up to 10-fold but resulted in mixed inhibition. The mutations I819L, Q923V, and Y925A also gave mixed inhibition but without a change in inhibitor affinity. These data, and the 9-fold loss of SCH28080 affinity in the C813T mutant, suggest that the binding domain for SCH28080 contains the surface between L809 in the M5-6 loop and C813 at the luminal end of M6, approximately two helical turns down from the ion binding region, where it blocks the normal ion access pathway. On the basis of a model of the Ca-ATPase in the E2 conformation (PDB entry 1kju), the mutants that change the nature of the kinetics are arranged on one side of M8 and on the adjacent side of the M5-6 loop and M6 itself. This suggests that mutations in this region modify the enzyme structure so that K+ can access the ion binding domain even with SCH28080 bound.     

Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.     

Overall Prevalence and Distribution of Knockdown Resistance (kdr) Mutations in Aedes aegypti from Mandalay Region,Myanmar

Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to insecticides. Although insecticide resistance has been suspected to be widespread in the natural population of Aedes aegypti in Myanmar, only limited information is currently available. The overall prevalence and distribution of kdr mutations was analyzed in Ae. aegypti from Mandalay areas, Myanmar. Sequence analysis of the VGSC in Ae. aegypti from Myanmar revealed amino acid mutations at 13 and 11 positions in domains II and III of VGSC, respectively. High frequencies of S989P (68.6%), V1016G (73.5%), and F1534C (40.1%) were found in domains II and III. T1520I was also found, but the frequency was low (8.1%). The frequency of S989P/V1016G was high (55.0%), and the frequencies of V1016G/F1534C and S989P/V1016G/F1534C were also high at 30.1% and 23.5%, respectively. Novel mutations in domain II (L963Q, M976I, V977A, M994T, L995F, V996M/A, D998N, V999A, N1013D, and F1020S) and domain III (K1514R, Y1523H, V1529A, F1534L, F1537S, V1546A, F1551S, G1581D, and K1584R) were also identified. These results collectively suggest that high frequencies of kdr mutations were identified in Myanmar Ae. aegypti, indicating a high level of insecticide resistance.