Histidine-rich Motif Research Articles

ATN1-related infantile developmental and epileptic encephalopathy responding to Ketogenic diet

BackgroundResearch has shown gene ATN1 to be associated with the nuclear receptor signaling. Its mutations in an evolutionarily conserved histidine-rich motif may cause CHEDDA, short for congenital hypotonia, epilepsy, developmental delay and digital anomalies, a recently identified neurodevelopmental syndrome that could evolve into developmental and epileptic encephalopathy (DEE). Up to date, there have been reported less than 20 cases, whose clinical features and treatment are worth in-depth exploring. MethodsThe clinical characteristics and genetic data of an infant with CHEDDA and further DEE were analyzed, who carried a de novo ATN1 variant identified by trio whole-exome sequencing. The alike patients with such a neurodevelopmental syndrome and epileptic seizures were reviewed on the literature. ResultsThe infant harboring a de novo missense mutation in ATN1 (c.3155A>C; p.His1052Pro) held almost all features of CHEDDA and presented with drug-resistant epileptic spasms, differing from one case previously reported with the same gene variant exhibiting milder seizures controlled easily. We further reviewed 11 CHEDDA patients with epileptic seizures in the literature and compared the correlation between abnormal cerebral structure and the incidence of intractable epilepsy among CHEDDA patients. Fortunately, this patient's seizures decreased remarkably after administering ketogenic diet (KD). ConclusionCHEDDA patients have significant phenotypic differences, especially in the epilepsy severity and their drug resistance, even if they carry the same mutation hotspot. Ketogenic diet and other treatments like Topiramate should be recommended for ATN1-related refractory epilepsy based on their regulation on expression of cation-chloride cotransporters and cellular hyperpolarization.

EP185: A novel, milder case of CHEDDA syndrome caused by a de novo variant outside of the canonical HX-motif of ATN1

Congenital hypotonia, epilepsy, developmental delay, and digital anomalies (CHEDDA) Syndrome is a neurodevelopmental disorder previously documented in over 17 unrelated individuals. The syndrome is caused by heterozygous variants in histidine-rich (HX) motif at exon 7 of ATN1. Other features of this syndrome include intellectual disability, severe motor disability with gait disturbance, feeding difficulties, hearing impairment, respiratory symptoms, and congenital malformations of the brain, heart, and genitourinary systems.

HRM and CRAC in MxIRT1 act as iron sensors to determine MxIRT1 vesicle-PM fusion and metal transport

ABSTRACT The IRON-REGULATED TRANSPORTER1 (IRT1) is critical for iron uptake in roots, and its exocytosis to the plasma membrane (PM) is regulated by detergent-resistant membranes. However, studies on IRT1 exocytosis and function in response to iron status are limited. Presently, we found that the histidine-rich motif (HRM) of MxIRT1 could bind to iron directly and HRM determined the delivery of MxIRT1 to the PM, after which the cholesterol recognition amino acid consensus (CRAC) motif-regulated MxIRT1 mediated metal transport. IMAC assay revealed that H192 was the vital site for HRM binding to Fe2+, and metal-binding activity was stopped after the deletion of HRM (MxIRT1∆HM) or in H192 site-directed mutants (H192A). MxIRT1∆HM or H192A in transgenic yeast and Arabidopsis failed to localize in the PM and displayed impaired iron absorption. In the PM, Y266 in CRAC was required for metal transport; Y266A transgenic Arabidopsis displayed the same root length, Cd2+ flux, and Fe concentration as Arabidopsis mutant irt1 under iron-deficient conditions. Therefore, H192 in HRM may be an iron sensor to regulate delivery of MxIRT1 vesicles to the PM after binding with iron; Y266 in CRAC acts as an iron sensor for active metal transport under iron-deficient conditions.

Metal transporter encoding gene families in Fabaceae: III. The zinc-iron permease (ZIP) gene family

In plants, Zinc and Iron are transported through the membrane by proteins belonging to Zinc-Iron permease (ZIP: ZRT/IRT-like Protein). In this work, the ZIP gene families were identified in the genome of five legume species. The results demonstrated that the ZIPs were belonged to a multigeneic family in each species including soybean (28 genes), Medicago truncalata (16 genes), chickpea (7 genes), pigeon pea (12 genes), and Lotus japonicus (15 genes). Each gene contained from one to twelve introns. ZIP proteins possessed a conserved histidine-rich motif. Most of these proteins contained eight putative transmembrane domains and were predicted to be localized in plasma membranes. The phylogeny analysis showed that the legume ZIPs were classified into four main groups, each of which includes many subgroups. The group I contained the ZIP members of five examined plants. Moreover, the phylogeny showed gene gain events (expansion) in group I and gene loss events in other groups. The gene expansion in group I is likely to have arisen mainly from recent duplication events of ZIP genes in the examined legume plants, after specialization. The expression analysis showed that all of ZIP genes were expressed in all of the examined tissues in L. japonicus. The expression level of ZIP members was not similar in different tissues of the plant. Some ZIP genes were predominantly expressed in certain tissues for most of the legume species investigated.

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately −7°C) and cold-acclimated (CA; leaf LT50 approximately −50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Ni(II) interaction with a peptide model of the human TLR4 ectodomain

Ni(II) stimulates innate immunity via the direct binding to human Toll Like Receptor 4 (hTLR4), the bacterial lypopolysaccharide receptor. The binding is specific for humans and causes nickel contact allergy. The protein sequence analysis of hTLR4 revealed that the ectodomain, the region supposed to coordinate the metal ions, contains a histidine-rich motif that is not conserved among all organisms. To elucidate the role of each histidine residue on the protein–nickel binding, we examined the formation of Ni(II) complexes with the model peptide NH2-FQHSNRKQMSERSVFRSRRNRIYRDISHTHTR-COO−, which encompasses the sequence 429–460 of hTLR4. The amino acid sequence of the peptide has been modified by the substitution of some selected lipophilic residues (Leu and Phe) with hydrophilic residues (Arg), aiming at increasing the peptide hydro solubility of the protein fragment. Potentiometric, ultraviolet-visible (UV–vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) measurements demonstrate that the non-conserved histidines in the ectodomain cooperate in metal coordination and consequently enable the activation of the molecular mechanism of nickel hypersensitivity reaction.

Identification and evolutionary relationships of partial gene sequences from dehydrin group in three species of cacti

Dehydrins or Group 2 Late Embryogenesis Abundant(LEA) proteins play an important role in the response and adaptationto different types of abiotic stresses such as droughts, high salinityand low temperatures. Using PCR techniques, we identified threegene fragments that encoded dehydrin-like proteins in three cactispecies Opuntia ficus-indica (OpfiDHN-like), Leuchtenbergia principis(LepDHN-like) and Mammillaria bombycina (MabDHN-like).Bioinformatic sequence analysis showed an identity between 96 and97% with the Opuntia streptacantha dehydrin 1 (OpsDHN1) gene,demonstrating that the amplified fragments corresponded to dehydrin-like gene sequences, and that the designed oligonucleotideswere effective for similar gene amplification in different cacti genera.Multiple OpfiDHN-like, LepDHN-like and MabDHN-like alignmentsshowed that they possessed three repetitions of the conservedK segment. Also, a histidine rich motif was found, which is believedto facilitate the binding of these proteins with metal ions that probablyevolved differently in the Opuntioidea and Cactoidea subfamiliesof the Cactaceae family. Bioinformatic tools demonstratedthat each of the three partial amino acid sequences corresponded toacidic, highly hydrophilic, and disordered protein fragments, whichare characteristics of dehydrin proteins. Phylogenetic analysis usingmaximum parsimony, indicated that cacti dehydrins-like proteinswere monophyletic, as well as those of other families.

Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif.

The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

Heavy Metal Binding and Accumulation of Genetically Engineered E. coli Harboring the CXXC Motif and Histidine Rich Motif Fusion Proteins

Heavy Metal Binding and Accumulation of Genetically Engineered E. coli Harboring the CXXC Motif and Histidine Rich Motif Fusion Proteins

Phylogenetic analysis of receptor FgfrL1 shows divergence of the C-terminal end in rodents.

FGFRL1 is a member of the fibroblast growth factor receptor (FGFR) family. Similar to the classical receptors FGFR1-FGFR4, it contains three extracellular Ig-like domains and a single transmembrane domain. However, it lacks the intracellular tyrosine kinase domain that would be required for signal transduction, but instead contains a short intracellular tail with a peculiar histidine-rich motif. This motif has been conserved during evolution from mollusks to echinoderms and vertebrates. Only the sequences of FgfrL1 from a few rodents diverge at the C-terminal region from the canonical sequence, as they appear to have suffered a frameshift mutation within the histidine-rich motif. This mutation is observed in mouse, rat and hamster, but not in the closely related rodents mole rat (Nannospalax) and jerboa (Jaculus), suggesting that it has occurred after branching of the Muridae and Cricetidae from the Dipodidae and Spalacidae. The consequence of the frameshift is a deletion of a few histidine residues and an extension of the C-terminus by about 40 unrelated amino acids. A similar frameshift mutation has also been observed in a human patient with a craniosynostosis syndrome as well as in several patients with colorectal cancer and bladder tumors, suggesting that the histidine-rich motif is prone to mutation. The reason why this motif was conserved during evolution in most species, but not in mice, is not clear.

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

A dehydrin-dehydrin interaction: the case of SK3 from Opuntia streptacantha.

Dehydrins belongs to a large group of highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins. It is well known that dehydrins are intrinsically disordered plant proteins that accumulate during the late stages of embryogenesis and in response to abiotic stresses; however, the molecular mechanisms by which their functions are carried out are still unclear. We have previously reported that transgenic Arabidopsis plants overexpressing an Opuntia streptacantha SK3 dehydrin (OpsDHN1) show enhanced tolerance to freezing stress. Herein, we show using a split-ubiquitin yeast two-hybrid system that OpsDHN1 dimerizes. We found that the deletion of regions containing K-segments and the histidine-rich region in the OpsDHN1 protein affects dimer formation. Not surprisingly, in silico protein sequence analysis suggests that OpsDHN1 is an intrinsically disordered protein, an observation that was confirmed by circular dichroism and gel filtration of the recombinantly expressed protein. The addition of zinc triggered the association of recombinantly expressed OpsDHN1 protein, likely through its histidine-rich motif. These data brings new insights about the molecular mechanism of the OpsDHN1 SK3-dehydrin.

A histidine-rich motif mediates mitochondrial localization of ZnT2 to modulate mitochondrial function

Female reproductive tissues such as mammary glands, ovaries, uterus, and placenta are phenotypically dynamic, requiring tight integration of bioenergetic and apoptotic mechanisms. Mitochondrial zinc (Zn) pools have emerged as a central player in regulating bioenergetics and apoptosis. Zn must first be imported into mitochondria to modulate mitochondrion-specific functions; however, mitochondrial Zn import mechanisms have not been identified. Here we documented that the Zn transporter ZnT2 is associated with the inner mitochondrial membrane and acts as an auxiliary Zn importer into mitochondria in mammary cells. We found that attenuation of ZnT2 expression significantly reduced mitochondrial Zn uptake and total mitochondrial Zn pools. Moreover, expression of a ZnT2-hemagglutinin (HA) fusion protein was localized to mitochondria and significantly increased Zn uptake and mitochondrial Zn pools, directly implicating ZnT2 in Zn import into mitochondria. Confocal microscopy of truncated and point mutants of ZnT2-green fluorescent protein (GFP) fusion proteins revealed a histidine-rich motif ((51)HHXH(54)) in the NH(2) terminus that is important for mitochondrial targeting of ZnT2. More importantly, the expansion of mitochondrial Zn pools by ZnT2 overexpression significantly reduced ATP biogenesis and mitochondrial oxidation concurrent with increased apoptosis, suggesting a functional role for ZnT2-mediated Zn import into mitochondria. These results identify the first Zn transporter directly associated with mitochondria and suggest that unique secretory tissues such as the mammary gland require novel mechanisms to modulate mitochondrion-specific functions.

Biology of FGFRL1, the fifth fibroblast growth factor receptor.

FGFRL1 (fibroblast growth factor receptor like 1) is the most recently discovered member of the FGFR family. It contains three extracellular Ig-like domains similar to the classical FGFRs, but it lacks the protein tyrosine kinase domain and instead contains a short intracellular tail with a peculiar histidine-rich motif. The gene for FGFRL1 is found in all metazoans from sea anemone to mammals. FGFRL1 binds to FGF ligands and heparin with high affinity. It exerts a negative effect on cell proliferation, but a positive effect on cell differentiation. Mice with a targeted deletion of the Fgfrl1 gene die perinatally due to alterations in their diaphragm. These mice also show bilateral kidney agenesis, suggesting an essential role for Fgfrl1 in kidney development. A human patient with a frameshift mutation exhibits craniosynostosis, arguing for an additional role of FGFRL1 during bone formation. FGFRL1 contributes to the complexity of the FGF signaling system.

Transmembrane topology of the mammalian Zip14 metal‐ion transporter

Zip14 belongs to the SLC39A family of metal‐ion transporters. Similar to general topology models of SLC39A proteins, Zip14 has been predicted to have 8 transmembrane domains with extracellular N‐ and C‐termini. In the present study, we used epitope tagging to investigate the topology of mouse Zip14. We inserted into Zip14 small antigenic FLAG peptides in the N‐terminus, C‐terminus, as well as the large extramembrane domain containing a histidine‐rich motif. The tagged proteins were expressed in HEK293 cells, and the polarity of the FLAG tags with respect to the plasma membrane were determined by immunofluorescence analysis of intact and permeabilized cells. Furthermore, glycosylation sites were identified by mutating each of the 5 potential N‐linked glycosylation sites, followed by Western blotting. Based on our analyses, combined with computer‐derived models, we conclude that mouse Zip14 has 7 transmembrane domains, an intracellular histidine‐rich region and c‐terminus, and an extracellular N‐terminus that is glycosylated at asparagines 52, 75, 85 and 100. The empirically determined structure of Zip14 will help to guide structure‐function analyses of this zinc/iron transporter.Grant Funding Source: national institute of health

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BackgroundFGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006).ResultsWe identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1.ConclusionThe FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

The diverse biofunctions of LIM domain proteins: determined by subcellular localization and protein—protein interaction

The LIM domain is a cysteine- and histidine-rich motif that has been proposed to direct protein-protein interactions. A diverse group of proteins containing LIM domains have been identified, which display various functions including gene regulation and cell fate determination, tumour formation and cytoskeleton organization. LIM domain proteins are distributed in both the nucleus and the cytoplasm, and they exert their functions through interactions with various protein partners.

A Histidine-rich Cluster Mediates the Ubiquitination and Degradation of the Human Zinc Transporter, hZIP4, and Protects against Zinc Cytotoxicity

Zinc is an essential nutrient. Genetic evidence for this nutritional requirement in humans is the zinc deficiency disease, acrodermatitis enteropathica. This disorder is caused by mutations in hZIP4 (SLC39A4), a zinc importer required for zinc uptake in enterocytes and other cell types. Studies in mice have demonstrated that levels of the mZIP4 mRNA are reduced by elevated dietary zinc, resulting in a decreased abundance of the ZIP4 protein at the plasma membrane. Moreover, studies in cultured cells have demonstrated that low micromolar concentrations of zinc stimulate the endocytosis of the mZIP4 protein resulting in a reduction in cellular zinc uptake. In this study, we demonstrate an additional level of hZIP4 regulation involving ubiquitination and degradation of this transporter in elevated zinc concentrations. Mutational analysis identified a cytoplasmic histidine-rich domain that was essential for ubiquitin-dependent degradation of ZIP4 and protection against zinc toxicity. However, this motif was dispensable for zinc-induced endocytosis. These findings indicate that ubiquitin-mediated degradation of the ZIP4 protein is critical for regulating zinc homeostasis in response to the upper tier of physiological zinc concentrations, via a process that is distinct from zinc-stimulated endocytosis.

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H 158WHD 161. To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.

Targeted mutagenesis of a fatty acid Δ 6-desaturase from Mucor rouxii: Role of amino acid residues adjacent to histidine-rich motif II

The amino acid residues serine at position 213 (S213) and lysine at position 218 (K218), which are present in close proximity to the histidine-rich motif II of Mucor rouxii fatty acid Δ 6-desaturase isoform II, were targeted for studying structure–function relationships using site-directed mutagenesis. The mutants were functionally characterized in a heterologous host, Saccharomyces cerevisiae. Substrate specificity and preference studies revealed that S213 and K218 are involved in substrate recognition. K218 plays a role in substrate preference by involvement in the binding of substrates, particularly C15–C18 monoene fatty acids. Modification of the M. rouxii Δ 6-desaturase therefore has potential in specifically altering substrate utilization for production of desired fatty acids.