HINT1 gene pathogenic variants: the most common cause of recessive hereditary motor and sensory neuropathies in Russian patients

Pathogenic variants in the HINT1 gene lead to hereditary axonopathy with neuromyotonia. However, many studies show that neuromyotonia may remain undiagnosed, while axonopathy is the major clinical finding. The most common cause of neuromyotonia and axonopathy, especially in patients of Slavic origin, is a c.110G>C (p.Arg37Pro) pathogenic variant in homozygous or compound heterozygous state. In this study, we analyzed a peripheral neuropathy caused by pathogenic variants in the HINT1 gene and evaluated its contribution to the hereditary neuropathy structure. The studied group included 1596 non-related families diagnosed with hereditary motor and sensory neuropathy (HMSN). The results show that HINT1 gene pathogenic variants make a significant contribution to the hereditary neuropathy epidemiology in Russian patients. They account for at least 1.9% of all HMSN cases and 9% of axonopathy cases. The most common HINT1 pathogenic variant in Russian patients is the c.110G>C (p.Arg37Pro) substitution. Its allelic frequency is 0.2% (95% CI 0.19–0.21%), carrier frequency is 1 in 250 people in Russian Federation, and the estimated disease incidence is 1 in 234,000 individuals. It was determined that the cause of this pathogenic variant’s prevalence is the founder effect.

     

Evidence that human histidine triad nucleotide binding protein 3 (Hint3) is a distinct branch of the histidine triad (HIT) superfamily

Human Hint3 (hHint3) has been classified as a member of the histidine triad nucleotide (Hint) binding protein subfamily. While Hint1 is ubiquitously expressed by both eukaryotes and prokaryotes, Hint3 is found only in eukaryotes. Previously, our laboratory has characterized and compared the aminoacyl-adenylate and nucleoside phosphoramidate hydrolase activity of hHint1 and Escherichia coli hinT. In this study, hHint3-1(Ala36) and its single nucleotide polymorphism, hHint3-2 (A36G variant), were cloned, overexpressed, and purified. Steady-state kinetic studies with a synthetic fluorogenic indolepropinoic acyl-adenylate (AIPA) and with a series of fluorogenic tryptamine nucleoside phosphoramidates revealed that hHint3-1 and hHint3-2 are adenylate and phosphoramidate hydrolases with apparent second-order rate constants (kcat/Km) ranging from 10(2) to 10(6) s(-1) M(-1). Unlike hHint1, hHint3-1 and hHint3-2 prefer AIPA over tryptamine adenosine phosphoramidate by factors of 33- and 16-fold, respectively. In general, hHint3s hydrolyze phosphoramidate 370- to 2000-fold less efficiently than hHint1. Substitution of the potential active-site nucleophile, His145, by Ala was shown to abolish the adenylate and phosphoramidate hydrolase activity for hHint3-1. However, 0.2-0.4% residual activity was observed for the H145A mutant of hHint3-2. Both hHint3-1 and hHint3-2 were found to hydrolyze lysyl-adenylate generated by human lysyl-tRNA synthetase (hLysRS) by proceeding through an adenylated protein intermediate. hLysRS-dependent labeling of hHint3-1 and hHint3-2 was found to depend on His145, which aligns with the His112 of the Hint1 active site. The extent of active-site His145-AMP labeling was shown to be similar to His112-AMP labeling of hHint1. In contrast to all previously characterized members of the histidine triad superfamily, which have been shown to exist exclusively as homodimers, wild type and the H145A of hHint3-1 were found to exist across a range of multimeric states, from dimers to octamers and even larger oligomers, while wild type and the H145A of hHint3-2 exist predominantly in a monomeric state. The differences in oligomeric state may be important in vivo, because unlike tetracysteine-tagged Hint1, which was found along linear arrays exclusively in the cytoplasm in transfected HeLa cells, tagged Hint3-1 and Hint3-2 were found as aggregates both in the cytosol and in the nucleus. Taken together, these results imply that while Hint3 and Hint1 prefer aminoacyl-adenylates as substrates and catalytically interact with aminoacyl-tRNA synthetases, the significant differences in phosphoramidase activity, oligomeric state, and cellular localization suggest that Hint3s should be placed in a distinct branch of the histidine triad superfamily.     

Highly conserved glycine 86 and arginine 87 residues contribute differently to the structure and activity of the mature HIV‐1 protease

The structural and functional role of conserved residue G86 in HIV‐1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PR_G86A and PR_G86S). While PR_G86S had undetectable catalytic activity, PR_G86A exhibited ～6000‐fold lower catalytic activity than PR. ¹H‐¹⁵N NMR correlation spectra revealed that PR_G86A and PR_G86S are dimeric, exhibiting dimer dissociation constants (K_d) of ～0.5 and ～3.2 μM, respectively, which are significantly lower than that seen for PR with R87K mutation (K_d > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PR_G86A and PR_G86S in the presence of active‐site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A conserved histidine in human DNLZ/HEP is required for stimulation of HSPA9 ATPase activity

The DNL-type zinc-finger protein DNLZ regulates the activity and solubility of the human mitochondrial chaperone HSPA9. To identify DNLZ residues that are critical for chaperone regulation, we carried out an alanine mutagenesis scan of charged residues in a W115I mutant of human DNLZ and assessed the effect of each mutation on interactions with HSPA9. All mutants analyzed promote the solubility of HSPA9 upon expression in Escherichia coli. However, binding studies examining the effect of DNLZ mutants on chaperone tryptophan fluorescence identified three mutations (R81A, H107A, and D111A) that decrease DNLZ binding affinity for nucleotide-free chaperone. In addition, ATPase measurements revealed that DNLZ–R81A and DNLZ–D111A both stimulate the catalytic activity HSPA9, whereas DNLZ–H107A does not elicit an increase in activity even when present at a concentration that is 10-fold higher than the level required for half-maximal stimulation by DNLZ. These findings implicate a conserved histidine as critical for DNLZ regulation of mitochondrial HSPA9 catalytic activity.     

Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val(97) of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277- and 1000-fold more efficient (k(cat)/K(m) values) than the V97E and V97D mutants, respectively. Adenylation of wild-type, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k(cat)/K(m) values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k(cat)/K(m)) of acylated AMP hydrolysis, but not maximal catalytic turnover (k(cat)), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.     

组氨酸三聚体核苷结合蛋白1和2与疾病相关的研究进展

组氨酸三聚体核苷结合蛋白(HINT)属于HIT蛋白超家族,HINT1和HINT2基因分别位于人类染色体5q31.2和9p11区域,编码产物分别是含126和163个氨基酸的蛋白,相对分子质量分别约14 kDa和17 kDa,二者之间有61％的序列同源性.HINT1在细胞中定位于细胞质和细胞核,HINT2定位于线粒体,二者...     

Nucleotide binding and dimerization at the chloroplast pre‐protein import receptor,atToc33, are not essential in vivo but do increase import efficiency

The atToc33 protein is one of several pre‐protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis‐related pre‐proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild‐type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159‐deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.     

Expression and purification of Arg196 and Lys272 mutants of mevalonate kinase from Methanococcus jannaschii

In microorganisms and plants, mevalonate kinase is involved in the biosynthesis of isoprenoid derivatives, one of the largest groups of natural products. We subcloned the gene of mevalonate kinase from Methanococcus jannaschii into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. A variety of mutant expression plasmids including pMMK(R196K), pMMK(R196Q), pMMK(R196V), pMMK(K272R), and pMMK(K272A) have been constructed using site-directed mutagenesis. The wild-type protein and mutants were overexpressed and purified with a nickel HiTrap chelating metal affinity column to homogeneity. CD spectroscopy of wild-type protein and mutants indicates that none of the above mutations induces significant secondary structural changes. The results from kinetic studies showed that Arg196 is an essential residue for the function of the enzyme. Kinetic studies of Lys272 mutants indicate that salt bridge Lys272-Glu14 plays an important role in maintaining the active site microenvironment that is essential for catalytic activity of the enzyme.     

Acute behavioral effects of nicotine in male and female HINT1 knockout mice

Human genetic association and brain expression studies, and mouse behavioral and molecular studies implicate a role for the histidine triad nucleotide‐binding protein 1 (HINT1) in schizophrenia, bipolar disorder, depression and anxiety. The high comorbidity between smoking and psychiatric disorders, schizophrenia in particular, is well established. Associations with schizophrenia and HINT1 are also sex specific, with effects more predominant in males; however, it is unknown if sex differences associated with the gene extend to other phenotypes. Thus, in this study, using a battery of behavioral tests, we elucidated the role of HINT1 in acute nicotine‐mediated behaviors using male and female HINT1 wild‐type (+/+) and knockout (?/?) mice. The results show that male HINT1 ?/? mice were less sensitive to acute nicotine‐induced antinociception in the tail‐flick, but not hot‐plate test. At low nicotine doses, male and female HINT1 ?/? mice were less sensitive to nicotine‐induced hypomotility, although the effect was more pronounced in females. Baseline differences in locomotor activity observed in male HINT1 +/+ and ?/? mice were absent in females. Nicotine did not produce an anxiolytic effect in male HINT1 ?/? mice, but rather an anxiogenic response. Diazepam also failed to induce an anxiolytic response in these mice, suggesting a general anxiety phenotype not specific to nicotine. Differences in anxiety‐like behavior were not observed in female mice. These results further support a role for HINT1 in nicotine‐mediated behaviors and suggest that alterations in the gene may have differential effects on phenotype in males and females.     

Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells

The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.     

NMR analysis of site-specific mutants of yeast phosphoglycerate kinase. An investigation of the triose-binding site

Site-specific mutants of yeast phosphoglycerate kinase have been produced in order to investigate the roles of the 'basic-patch' residues, arginine 168 and histidine 170. The fully-conserved residue, arginine 168, has been replaced with a lysine (R168K) and a methionine (R168M) residue, while the non-conserved histidine 170 has been replaced with an aspartate (H170D). Comparison of the 500-MHz 1H-NMR spectra of the mutant proteins with that of wild-type phosphoglycerate kinase shows that the overall fold of the mutants remains essentially unaltered from that of the native enzyme. Results of NOE experiments indicate that there are only very minor changes in structure in the vicinity of the mutations. These mutations have also led to firm sequence-specific resonance assignments to histidines 62, 167 and 170. NMR studies of 3-phosphoglycerate binding show that decreasing the positive charge in the sequence 168-170 reduces the binding of this substrate (by about 15-fold and 4-fold for mutants R168M and H170D respectively). Mutant R168K binds 3-phosphoglycerate with an affinity about twofold less than that of the native enzyme. Significantly, the activity of mutant H170D, measured at saturating substrate concentrations, is unchanged from that of the wild-type enzyme. This indicates that this residue is not of major importance in the binding or reaction of 3-phosphoglycerate. The observation is in agreement with results obtained for the wild-type enzyme, which indicate that 3-phosphoglycerate interacts most strongly with histidine 62 and least strongly with histidine 170, as would be predicted from the X-ray crystal structure. Substitution of positively charged arginine 168 with neutral methionine (or positively charged lysine) does not cause a detectable change in the pKa values of the neighbouring histidine groups, in as much as they remain below 3. The results reported here indicate that the observed reduction in catalytic efficiency relates less to direct electrostatic effects than to the mutants' inability to undergo 3-phosphoglycerate-induced conformational changes.     

Biochemical basis for the dominant inheritance of hypermethioninemia associated with the R264H mutation of the MAT1A gene. A monomeric methionine adenosyltransferase with tripolyphosphatase activity

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields a MAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PP(i). Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.     

Identification of amino acid residues essential to the activity of lyase CpcT1 from Nostoc sp. PCC7120

The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1.     

Functional characterization of MODY2 mutations in the nuclear export signal of glucokinase

Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence.Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning.In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.     

Mutagenesis of the catalytic subunit of rabbit muscle protein phosphatase-1

We have generated site-directed mutants of the catalytic subunit of rabbit muscle ppase-1. Since it is known that ppase-1 and ppase-2A are highly susceptible to inactivation by sulfhydryl reagents, we have mutagenized the six cysteine residues conserved between these two enzymes to serines. The six mutants were purified to near homogeneity by affinity chromatography on inhibitor-2-Sepharose and characterized. All six exhibited enzymatic activity. These results indicate that the catalytic mechanism of ppase-1 is different from that of the protein tyrosine phosphatases which involve a cysteinyl phosphate intermediate.     

Characterization of Mutants of Human Small Heat Shock Protein HspB1 Carrying Replacements in the N-Terminal Domain and Associated with Hereditary Motor Neuron Diseases

Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1.     

Characterization of Escherichia coli ATP synthase beta-subunit mutations using a chromosomal deletion strain.

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.     

Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins,including nitrated proteins

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.