Histidine Triad Nucleotide-binding Protein Research Articles

Hint1 gene deficiency enhances the supraspinal nociceptive sensitivity in mice

IntroductionPrevious studies have indicated a possible role of histidine triad nucleotide‐binding protein 1 (HINT1) on sustaining the regulatory crosstalk of N‐methyl‐D‐aspartate acid glutamate receptors (NMDARs) and G‐protein‐coupled receptors (GPCRs) such as the μ‐opioid receptor (MOR). Both receptors are present in the midbrain periaqueductal gray neurons, an area that plays a central role in the supraspinal antinociceptive process.MethodsIn the present study, a battery of pain‐related behavioral experiments was applied to Hint1 knockout, heterozygous and wild‐type mice. Both the male and female mice were investigated to assess the differences between genders.Results Hint1−/− mice presented significant shorter latency at 50°C in both male and female in hot plate test while no significant difference was found in tail filck test. In Von Frey hairs test Hint1−/− mice were more sensitive than Hint1+/+ mice, presenting a lower withdrawal threshold and enhanced relative frequency of paw withdrawal. The average flinches and licking time of Hint1−/− mice were more than that of Hint1+/+ mice in formalin test.ConclusionThe absence of Hint1 gene‐enhanced supraspinal nociceptive sensitivity in mice, including thermal, mechanical and inflammatory hyperalgesia. Meanwhile, there was no certain evidence indicating the haploinsufficiency and gender differences of Hint1 gene in pain‐related behaviors.

Construction of a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene and its effects on apoptosis and autophagy of A375 melanoma cells

Objective To construct a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene, to investigate its expression in A375 melanoma cells after transfection, and to evaluate its effects on apoptosis and autophagy of A375 cells. Methods The PKCI-1/HINT1 gene sequence was amplified by reverse transcription PCR (RT-PCR)with total RNA extracted from A375 cells as the template, then inserted into the eukaryotic expression plasmid PCDNA3.1(+)to construct a recombinant plasmid, PCDNA3.1(+)-PKCI-1/HINT1. Some A375 cells were classified into two groups to be transiently transfected with the recombinant plasmid (PCDNA3.1 (+)-PKCI-1/HINT1 group)or the empty plasmid PCDNA3.1(+)(control group). After additional 48-hour culture, RT-PCR and Western blot analysis were performed to quantify the mRNA and protein expressions of PKCI-1/HINT1 respectively, Hoechst 33342 staining was conducted to detect apoptosis of A375 cells, Western blot analysis to detect the expressions of intracellular caspase-3 and autophagyassociated protein beclin1, and cell autophagy was observed by using the green fluorescent protein (GFP)microtubule-associated protein 1 light chain 3 (LC3)labelling method combined with confocal laser scanning microscopy. Methyl thiazolyl tetrazolium (MTT)assay was performed to evaluate the proliferative activity of A375 cells at 24, 48, 72 and 96 hours after transfection. Results Enzyme digestion and sequencing analysis confirmed that the eukaryotic expression plasmid PCDNA3.1(+)-PKCI-1/HINT1 was successfully constructed and effectively expressed in the transfected A375 cells. MTT assay showed that PKCI-1/HINT1 could obviously inhibit the proliferation of A375 cells, and the number of live cells was decreased by 17.0%, 25.6% and 29.4% in the PCDNA3.1(+)-PKCI-1/HINT1 group at 48, 72 and 96 hours, respectively, compared with the control group (all P < 0.05). Hoechest 33258 staining revealed that PKCI-1/HINT1 could promote the formation of apoptotic bodies in A375 cells. Confocal laser scanning microscopy demonstrated that the overexpression of PKCI-1/HINT1 increased GFP-LC3 puncta formation in A375 cells. In addition, Western blot analysis indicated that PKCI-1/HINT1 upregulated the protein expressions of caspase-3 and beclin1 in A375 cells. Conclusions The eukaryotic expression plasmid PCDNA3.1 (+ )-PKCI-1/HINT1 was successfully constructed, and PKCI-1/HINT1 could be effectively expressed in A375 cells. High-level expression of PKCI-1/HINT1 could suppress cellular proliferation, promote apoptosis, and induce autophagy, of A375 cells. Key words: Melanoma; Cell line, tumor; Apoptosis; Autophagy; PKCI-1/HINT1

Crystallographic studies of the complex of human HINT1 protein with a non-hydrolyzable analog of Ap4A

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine triad proteins superfamily. HINT1 plays an important role in various biological processes, and it has been found in many species. Here, we report the first structure (at a 2.34Å resolution) of a complex of human HINT1 with a non-hydrolyzable analog of an Ap4A dinucleotide, containing bis-phosphorothioated glycerol mimicking a polyphosphate chain, obtained from a primitive monoclinic space group P21 crystal. In addition, the apo form of hHINT1 at the space group P21 refined to 1.92Å is reported for comparative studies.

The Effect of Weight Loss on the Muscle Proteome in the Damara, Dorper and Australian Merino Ovine Breeds.

Seasonal Weight Loss (SWL) is an important constraint, limiting animal production in the Tropics and the Mediterranean. As a result, the study of physiological and biochemical mechanisms by which domestic animal breeds respond to SWL is important to those interested in animal breeding and the improvement thereof. To that end, the study of the proteome has been instrumental in gathering important information on physiological mechanisms, including those underlying SWL. In spite of that, little information is available concerning physiological mechanisms of SWL in production animals. The objective of this study was to determine differential protein expression in the muscle of three different breeds of sheep, the Australian Merino, the Dorper and the Damara, each showing different levels of tolerance to weight loss (low, medium and high, respectively). Per breed, two experimental groups were established, one labeled “Growth” and the other labeled “Restricted.” After forty-two days of dietary treatment, all animals were euthanized. Muscle samples were then taken. Total protein was extracted from the muscle, then quantified and two-dimensional gel electrophoresis were conducted using 24 cm pH 3–10 immobiline dry strips and colloidal coomassie staining. Gels were analyzed using Samespots® software and spots of interest were in-gel digested with trypsin. The isolated proteins were identified using MALDI-TOF/TOF. Results indicated relevant differences between breeds; several proteins are suggested as putative biomarkers of tolerance to weight loss: Desmin, Troponin T, Phosphoglucomutase and the Histidine Triad nucleotide-binding protein 1. This information is of relevance to and of possible use in selection programs aiming towards ruminant animal production in regions prone to droughts and weight loss.

Hint1 Up-Regulates IκBα by Targeting the β-TrCP Subunit of SCF E3 Ligase in Human Hepatocellular Carcinoma Cells.

There is increasing evidence that histidine triad nucleotide-binding protein 1 (HINT1) is a novel tumor suppressor. In the present study, we investigated the mechanism by which HINT1 promotes the stability of inhibitor of NF-κB α (IκBα) in the cytoplasm of hepatocellular carcinoma (HCC) cells, which was observed in our previous study (Wang et al. in Int J Cancer 124:1526-1534, 2009). We examined HINT1 and IκBα expression in HCC cell lines and determined the effect of HINT1 overexpression and knockdown on IκBα protein and mRNA expression in these cell lines. Then, ubiquitination assays were performed to investigate the effects of HINT1 expression plasmid transfection on IκBα ubiquitination. Next, the interaction between HINT1 and β-TrCP was investigated in immunoprecipitation and immunofluorescence assays. Our data showed that increased HINT1 expression in HepG2 and SMMC7702 cells markedly increased IκBα protein levels, while decreased HINT1 expression markedly decreased them. Overexpression or knockdown of HINT1 did not alter the transcription of IκBα, but HINT1 inhibited proteasomal IκBα degradation and reduced its ubiquitination levels. This inhibition might occur because HINT1 is a component of the SCF(β-TrCP) E3 ligase, which is responsible for IκBα ubiquitination and degradation. This study provides new evidence that HINT1 is a regulator of IκBα through SCF(β-TrCP) E3 ligase. These findings help to clarify the mechanism underlying the anticancer effects of HINT1.

PKC GAMMA-MEDIATED SIGNALING AND SCHIZOPHRENIA

Schizophrenia is a complicated mental disorder. Although multiple hypotheses have been proposed to interpret the molecular mechanisms underlying the pathogenesis of the disease, the key pathological process is yet blurry. Early clinical studies have implicated a role of the histidine triad nucleotide-binding protein-1 (Hint1) in the pathogenesis of schizophrenia. A most recent investigation by Zhang et al has defined the activity of protein PKC gamma (PKC𝛾) in the brain using Hint1-knockout animal model. Their finding that Hint1-deficiency causes a compromised PKC𝛾 signaling in the brain may shed a new light on the glutamate hypothesis of schizophrenia.

The ON:OFF switch, σ1R-HINT1 protein, controls GPCR-NMDA receptor cross-regulation: implications in neurological disorders.

In the brain, the histidine triad nucleotide-binding protein 1 (HINT1) and sigma 1 receptors (σ1Rs) coordinate the activity of certain G-protein coupled receptors (GPCRs) with that of glutamate N-methyl-D-aspartate receptors (NMDARs). To determine the role of HINT1-σ1R in the plasticity of GPCR-NMDAR interactions, substances acting at MOR, cannabinoid CB1 receptor, NMDAR and σ1R were injected into mice, and their effects were evaluated through in vivo, ex vivo, and in vitro assays. It was observed that HINT1 protein binds to GPCRs and NMDAR NR1 subunits in a calcium-independent manner, whereas σ1R binding to these proteins increases in the presence of calcium. In this scenario, σ1R agonists keep HINT1 at the GPCR and stimulate GPCR-NMDAR interaction, whereas σ1R antagonists transfer HINT1 to NR1 subunits and disengage both receptors. This regulation is lost in σ1R-/- mice, where HINT1 proteins mostly associate with NMDARs, and GPCRs are physically and functionally disconnected from NMDARs. In HINT1-/- mice, ischemia produces low NMDAR-mediated brain damage, suggesting that several different GPCRs enhance glutamate excitotoxicity via HINT1-σ1R. Thus, several GPCRs associate with NMDARs by a dynamic process under the physiological control of HINT1 proteins and σ1Rs. The NMDAR-HINT1-σ1R complex deserves attention because it offers new therapeutic opportunities.

Hint1 knockout results in a compromised activation of protein kinase C gamma in the brain

Previous studies have implicated a role of the histidine triad nucleotide-binding protein 1 (Hint1) in the pathogenesis of schizophrenia. Protein kinase C gamma (PKCγ) could be potentially involved in the Hint1-implicated pathogenesis since PKCγ was identified as a Hint1 interacting protein. Recently, a debate was brought forward from the understanding how Hint1 affects the expression and activity of PKCγ in the brain. In the present study, we use Hint1 knockout mice and biochemical analysis to define the effect of Hint1 on protein PKCγ. Our data reveal that Hint1-deficiency in mouse brains led to increased protein levels of PKCγ in the cortex and hippocampus, the striatum and thalamus and amygdala. Without stimulation, PKCγ protein in Hint1-deficient brain displayed a basal activity that was reflected by control-leveled phosphorylations of PKCγ T514 and T674 at its kinase domain. Upon psycho-stimulation, both sites of PKCγ T514 and T674 were activated in these brain structures via phosphorylation; however, the phosphorylation level at the site of PKCγ T674 apparently attenuated in Hint1-deficient mice compared to wild-type control. Thus, we conclude that Hint1 deficiency leads to an increased protein level of PKCγ in the brain and a compromised activation response of PKCγ upon stimulation. These findings suggest an inhibitory role of Hint1 on the protein PKCγ in the brain and an impaired PKCγ-mediated phosphorylation signal in Hint1-deficient neuron.

Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5'-phosphoramidates and nucleoside 5'-phosphosulfates to generate nucleoside 5'-phosphorofluoridates.

Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20times and human Hint2 40times more slowly than human Fhit.

Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.

CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

The Intracellular Domain of Teneurin-1 Induces the Activity of Microphthalmia-associated Transcription Factor (MITF) by Binding to Transcriptional Repressor HINT1

Teneurins are large type II transmembrane proteins that are necessary for the normal development of the CNS. Although many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus, where it can influence gene transcription. Because teneurin ICDs do not contain any intrinsic DNA binding sequences, interaction partners are required to affect transcription. Here, we identified histidine triad nucleotide binding protein 1 (HINT1) as a human teneurin-1 ICD interaction partner in a yeast two-hybrid screen. This interaction was confirmed in human cells, where HINT1 is known to inhibit the transcription of target genes by directly binding to transcription factors at the promoter. In a whole transcriptome analysis of BS149 glioblastoma cells overexpressing the teneurin-1 ICD, several microphthalmia-associated transcription factor (MITF) target genes were found to be up-regulated. Directly comparing the transcriptomes of MITF versus TEN1-ICD-overexpressing BS149 cells revealed 42 co-regulated genes, including glycoprotein non-metastatic b (GPNMB). Using real-time quantitative PCR to detect endogenous GPNMB expression upon overexpression of MITF and HINT1 as well as promoter reporter assays using GPNMB promoter constructs, we could demonstrate that the teneurin-1 ICD binds HINT1, thus switching on MITF-dependent transcription of GPNMB.

The σ1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA receptor negative control.

Aims: The in vivo pharmacology of the sigma 1 receptor (σ1R) is certainly complex; however, σ1R antagonists are of therapeutic interest, because they enhance mu-opioid receptor (MOR)-mediated antinociception and reduce neuropathic pain. Thus, we investigated whether the σ1R is involved in the negative control that glutamate N-methyl-d-aspartate acid receptors (NMDARs) exert on opioid antinociception. Results: The MOR C terminus carries the histidine triad nucleotide-binding protein 1 (HINT1) coupled to the regulator of G-protein signaling RGSZ2-neural nitric oxide synthase assembly. Activated MORs stimulate the production of nitric oxide (NO), and the redox zinc switch RGSZ2 converts this signal into free zinc ions that are required to recruit the redox sensor PKCγ to HINT1 proteins. Then, PKCγ impairs HINT1-RGSZ2 association and enables σ1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. The inhibition of NOS or the absence of σ1Rs prevents HINT1-PKCγ interaction, and MOR-NMDAR cross-regulation fails. The σ1R antagonists transitorily remove the binding of σ1Rs to NR1 subunits, facilitate the entrance of negative regulators of NMDARs, likely Ca2+-CaM, and prevent NR1 interaction with HINT1, thereby impairing the negative feedback of glutamate on opioid analgesia. Innovation: A redox-regulated process situates MOR signaling under NMDAR control, and in this context, the σ1R binds to the cytosolic C terminal region of the NMDAR NR1 subunit. Conclusion: The σ1R antagonists enhance opioid analgesia in naïve mice by releasing MORs from the negative influence of NMDARs, and they also reset antinociception in morphine tolerant animals. Moreover, σ1R antagonists alleviate neuropathic pain, probably by driving the inhibition of up-regulated NMDARs. Antioxid. Redox Signal. 22, 799–818.

Differential proteomic analysis of the anti-depressive effects of oleamide in a rat chronic mild stress model of depression

Depression is a complex psychiatric disorder, and its etiology and pathophysiology are not completely understood. Depression involves changes in many biogenic amine, neuropeptide, and oxidative systems, as well as alterations in neuroendocrine function and immune-inflammatory pathways. Oleamide is a fatty amide which exhibits pharmacological effects leading to hypnosis, sedation, and anti-anxiety effects. In the present study, the chronic mild stress (CMS) model was used to investigate the antidepressant-like activity of oleamide. Rats were exposed to 10weeks of CMS or control conditions and were then subsequently treated with 2weeks of daily oleamide (5mg/kg, i.p.), fluoxetine (10mg/kg, i.p.), or vehicle. Protein extracts from the hippocampus were then collected, and hippocampal maps were generated by way of two-dimensional gel electrophoresis (2-DE). Altered proteins induced by CMS and oleamide were identified through mass spectrometry and database searches. Compared to the control group, the CMS rats exhibited significantly less body weight gain and decreased sucrose consumption. Treatment with oleamide caused a reversal of the CMS-induced deficit in sucrose consumption. In the proteomic analysis, 12 protein spots were selected and identified. CMS increased the levels of adenylate kinase isoenzyme 1 (AK1), nucleoside diphosphate kinase B (NDKB), histidine triad nucleotide-binding protein 1 (HINT1), acyl-protein thioesterase 2 (APT-2), and glutathione S-transferase A4 (GSTA4). Compared to the CMS samples, seven spots changed significantly following treatment with oleamide, including GSTA4, glutathione S-transferase A6 (GSTA6), GTP-binding nuclear protein Ran (Ran-GTP), ATP synthase subunit d, transgelin-3, small ubiquitin-related modifier 2 (SUMO2), and eukaryotic translation initiation factor 5A-1 (eIF5A1). Of these seven proteins, the level of eIF5A1 was up-regulated, whereas the remaining proteins were down-regulated. In conclusion, oleamide has antidepressant-like properties in the CMS rat model. The identification of proteins altered by CMS and oleamide treatment provides support for targeting these proteins in the development of novel therapies for depression.

Overexpression of Zm-HINT1 in Arabidopsis thaliana enhances resistance to Fusarium graminearum

Histidine triad nucleotide binding protein 1(HINT1) plays an important role in many biological processes especially in cell biology, and they have been found in a wide variety of species. However, the functional attributes of HINT1 homologues in plants have not yet been reported. We have previously isolated the full-length cDNA of an HINT1 homologue from maize (Zea mays L.), designated as Zm-HINT1. Subcellular localization was analysed after particle-mediated transient transformation of onion epidermal cells. The result revealed that Zm-HINT1 was targeted to the nucleus. Based on real-time PCR analysis, the expression level of Zm-HINT1 increased significantly after salicylic acid or jasmonic acid treatments. To further investigate the potential physiological role of Zm-HINT1, a binary vector was constructed for Zm-HINT1 synthesis and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Transgenic Zm-HINT1 plants showed increased resistance to Fusarium graminearum compared to the wild type and control plants. Futhermore, real-time PCR results revealed that the pathogen defense genes pathogenesis-related 4 (PR-4) and a peroxidase were upregulated by Zm-HINT1 overexpression. Thus, our data demonstrated that Zm-HINT1 induced antifungal activity against Fusarium graminearum and it may be useful for crop breeding in improving biotic stress tolerance.

HINT1 protein: A new therapeutic target to enhance opioid antinociception and block mechanical allodynia

In the nervous system, the glutamate N-methyl-d-aspartate receptor (NMDAR) restricts the activity of the mu-opioid receptor (MOR). Both receptors are present in midbrain periaqueductal grey (PAG) neurons, an area that plays a central role in the supraspinal antinociceptive effects of opioids. The cross-talk that occurs between these receptors is sustained by the MOR-associated histidine triad nucleotide binding protein 1 (HINT1), which displays nucleoside phosphoramidase and acyl-AMP hydrolase activity. Here we report that the inhibitor of HINT1 enzymatic activity guanosine-5′-tryptamine carbamate (TpGc) significantly enhanced morphine antinociception while preventing the development of tolerance. At the molecular level, TpGc reduced the capacity of MORs to recruit NMDAR activity to negatively regulate opioid signaling. In mice suffering from chronic constriction injury concurrent with increased NMDAR activity, a single intracerebroventricular administration of TpGc attenuated NMDAR function and alleviated mechanical allodynia for several days. These data suggest a potential therapeutic role for HINT1 inhibitors in the clinical management of acute and neuropathic pain.

Unraveling the genetic landscape of autosomal recessive Charcot-Marie-Tooth neuropathies using a homozygosity mapping approach.

Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes (ganglioside-induced differentiation protein 1—GDAP1, SH3 domain and tetratricopeptide repeats-containing protein 2—SH3TC2, histidine-triad nucleotide binding protein 1—HINT1) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 22% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.3% patients. Apart from a newly described founder mutation in GDAP1, the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.

Interactions of cellular histidine triad nucleotide binding protein 1 with nucleosides 5′-O-monophosphorothioate and their derivatives — Implication for desulfuration process in the cell

BackgroundOne of the activities of histidine triad nucleotide-binding protein 1 (Hint1) under in vitro conditions is the conversion of nucleoside 5′-O-phosphorothioate (NMPS) to its 5′-O-phosphate (NMP), which is accompanied by the release of hydrogen sulfide. MethodsNon-hydrolyzable derivatives of AMPS and dCMPS, each containing the residue able to form a covalent bond in nucleic acid–protein complexes via photocrosslinking (at 308nm), were applied at the complexing experiments with recombinant and cellular Hint1.The cellular lysates prepared after RNAi-mediated knockdown of Hint1 were incubated with AMPS and the level of desulfuration was measured. ResultsRecombinant Hint1 and Hint1 present in the cellular lysate of A549 cells, formed complexes with the used substrate analogs.Computer modeling experiments, in which the ligand was docked at the binding pocket, confirmed that direct interactions between Hint1 and the screened analogs are possible.Using RNAi technology, we demonstrated lowered levels of AMPS substrate desulfuration in reactions that employed the cell lysates with a reduced Hint1 level. ConclusionsThe enzymatic conversion of AMPS to AMP occurred with the participation of cellular Hint1, the protein, which is present in all organisms. General significanceThe intracellular Hint1 could be responsible for the in vivo desulfuration of nucleosides-5′-monophosphorothioate, thus it can contribute to the phosphorothioate oligonucleotides metabolism. H2S released during this process may participate in several physiological processes, thus NMPSs can be precursors/donors of H2S in vivo and can be used to study the effects of this gas in biological systems. Moreover, the controlled delivery of (d)NMPSs into cells may be of medicinal utility.

HINT1 peptide/Hsp70 complex induces NK-cell-dependent immunoregulation in a model of autoimmune demyelination.

Heat shock proteins (Hsps) interact with the immune system and have been shown to contribute to immunoregulation. As efficient chaperones, Hsps bind many peptides and these complexes have many yet-to-be-clarified functions. We have shown that Hsp70 is complexed within the mouse CNS with peptide CLAFHDISPQAPTHFLVIPK derived from histidine triad nucleotide-binding protein-1 (HINT1₃₈₋₅₇/Hsp70). Only this complex, in contrast to other peptides complexed with Hsp70, was able to prevent experimental autoimmune encephalomyelitis (EAE) by induction of immunoregulatory mechanisms dependent on NK cells. Pretreatment of proteolipid protein peptide ₁₃₉₋₁₅₁(PLP₁₃₉₋₁₅₁) sensitized SJL/J mice with HINT1₃₈₋₅₇/Hsp70 prevented the development of EAE, suppressed PLP₁₃₉₋₁₅₁-induced T-cell proliferation, and blocked secretion of IL-17. HINT1₃₈₋₅₇ /Hsp70 stimulation of NK cells depended on synergistic activation of two NK-cell receptors, CD94 and NKG2D. NK cells with depleted CD94 or with blocked NKG2D did not inhibit PLP₁₃₉₋₁₅₁-induced spleen cell (SC) proliferation. The HINT1₃₈₋₅₇/Hsp70 complex enhanced surface expression of the NKG2D ligand-H60. Downstream signaling of CD94 and NKG2D converged at the adaptor proteins DAP10 and DAP12, and in response to HINT1₃₈₋₅₇ /Hsp70 stimulation, expression of DAP10 and DAP12 was significantly increased in NK cells. Thus, we have shown that the HINT1₃₈₋₅₇ /Hsp70 complex affects NK-cell function by enhancing NK-cell-dependent immunoregulation in the EAE model of autoimmune demyelination.

Histidine triad nucleotide-binding protein 1 and human diseases

Histidine triad nucleotide-binding protein 1 (HINT1) is a member of a superfamily of histidine triad proteins named by the conserved nucleotide-binding motif histidine-x-histidine-x-histidine-xx, in which x represents hydrophobic amino acid. HINT1 is implicated in pathological progress of many human diseases including cancer and schizophrenia; however, little is known about the essential role and pathological consequences of HINT1 in cellular physiology and diseases. Therefore, we summarize the structure, distribution, and physiological function of HINT1 in cells and tissues as well as the correlation between HINT1 and human diseases.

Lack of neuropathy-related phenotypes in hint1 knockout mice.

Mutations in HINT1, the gene encoding histidine triad nucleotide-binding protein 1 (HINT1), cause a recessively inherited peripheral neuropathy that primarily involves motor dysfunction and is usually associated with neuromyotonia (i.e. prolonged muscle contraction resulting from hyperexcitability of peripheral nerves). Because these mutations are hypothesized to cause loss of function, we analyzed Hint1 knockout mice for their relevance as a disease model. Mice lacking Hint1 appeared normal and yielded normal behavioral test results or motor performance, although they moved more slowly and for a smaller fraction of time in an open-field arena than wild-type mice. Muscles, neuromuscular junctions, and nodes of Ranvier were anatomically normal and did not show evidence of degeneration or regeneration. Axon numbers and myelination in peripheral nerves were normal at ages 4 and 13 months. Axons were slightly smaller than those in wild-type mice at age 4 months, but this did not cause a decrease in conduction velocity, and no differences in axon diameters were detected at 13 months. With electromyography, we were unable to detect neuromyotonia even after using supraphysiologic stimuli and stressors such as reduced temperature or 3,4-diaminopyridine to block potassium channels. Therefore, we conclude that Hint1 knockout mice may be useful for studying the biochemical activities of HINT1, but these mice do not provide a disease model or a means for investigating the basis of HINT1-associated neuropathy and neuromyotonia.