Mutation Of Tyr Research Articles

A missense mutation in the tyrosinase gene explains acromelanism in domesticated canaries.

Acromelanism is a form of albinism observed in several vertebrate species. In mammals, acromelanism is known to be caused by mutations in the tyrosinase gene (TYR) that induce a temperature-sensitive behavior of melanin synthesis, resulting in a characteristic hair color gradient. In birds, several phenotypes consistent with acromelanism have been reported, but their genetic basis remains unknown. This study aimed to identify the genetic basis of an acromelanistic phenotype in domesticated canaries known as pearl and test whether it is caused by the same molecular mechanism described for mammals. To do this, we compared the genomes of pearl and non-pearl canaries and searched for potentially causative genetic mutations. Our results suggest that the pearl phenotype is caused by a mutation in the TYR gene encoding a TYR-P45H missense substitution. Our findings further suggest that reports of acromelanism in other bird species might be explained by TYR mutations.

Identifying genetic defects in oculocutaneous albinism patients of West Bengal, Eastern India.

Oculocutaneous albinism (OCA) is a congenital heterogeneous group of autosomal recessive disorders characterized by the absence or loss of melanin in the skin, eyes and hair of the affected individuals. Based on the mutated gene, OCA has been classified into eight sub-types (OCA1-8) with overlapping clinical phenotypes. Mutations in the TYR gene cause OCA1, the most prevalent OCA worldwide including India. Mutations in OCA2 and SLC45A2, both of which regulate melanosomal pH that is critical to TYR activity, cause OCA2 and OCA4 respectively, the other common OCA subtypes in India. In the present study, we have included 54 OCA-affected cases from 41 unrelated families representing 16 different marriage/ethnic groups from 17 districts of West Bengal, India. We pursued a PCR-sequencing based approach followed by bioinformatic analysis to identify mutations in TYR, OCA2 and SLC45A2 genes. Mutations were detected in 27 of the 54 (50%) OCA patients from 18 unrelated families, representing 9 different marriage/ethnic groups from 11 districts of West Bengal. Three TYR variants: NM_000372.4: c.391 A > G, NP_000363.1: p. Lys131Glu; NM_000372.4: c.1037G > T; NP_000363.1: p. Gly346Val, NM_000372.4: c.715 C > T; NP_000363.1:p.Arg239Trp was identified for the first time in Eastern Indian OCA cases. A novel nonsense variant: NM_016180.5: c.389T > A, NP_057264.4: p. Leu130* and a novel synonymous variation NM_016180.5: c.1092 A > G; NP_057264.4: p.364E = were identified in SLC45A2. Additionally, NM_016180.5: c.904A > T; NP_057264.4: p. Thre302Ser was identified for the first time in any Eastern Indian OCA case. We identified 2 previously reported mutations in OCA2. In concordance with previous reports, NM_000372.4: c.832C > T, NP_000363.1: p. (Arg278*) was the commonest TYR mutation. The results of our study enrich the mutational spectrum of the known OCA causing genes in Eastern India, which would facilitate accurate diagnosis, familial screening, carrier detection and containment of the disease load.

Nystagmus in the B6(CG)Tyr(c-2J)/J Albino Mouse: A Functional and RNA-Seq Analysis.

Infantile nystagmus syndrome (INS) is a gaze-holding disorder characterized by conjugate, uncontrolled eye oscillations that can result in significant visual acuity loss. INS is often associated with albinism, but the mechanism is unclear. Albino mice have nystagmus; however, a pigmented mouse with a tyr mutation making it phenotypically albino, the B6(CG)-Tyr(c-2J)/J (B6 albino), had not been tested. We tested optokinetic response (OKR) in B6 albino and control mice. RNA-Seq was performed on extraocular muscles (EOM), tibialis anterior (TA) muscle, abducens (CN6), and oculomotor (CN3) neurons to uncover molecular differences that may contribute to nystagmus. OKR was measured using an ISCAN system. RNA was isolated from four tissues to identify differentially expressed genes and validated with qPCR and immunohistochemistry. Ingenuity pathway analyses identified top biological pathways. All B6 albino mice tested had nystagmus. Differential RNA expression analysis showed 383 genes differentially expressed in EOM, 70 in CN3, 20 in CN6, and 639 in the TA. Two genes were differentially expressed in all four tissues: wdfy1 and nnt. Differences were validated by qPCR and immunostaining. The tyr mutation in B6 albino mice, genotypically pigmented and phenotypically albino, is sufficient to result in spontaneous nystagmus. The two genes with decreased expression in the B6 albino tissues examined, wdfy1 and nnt, have been implicated in mitochondrial dysfunction and stem cell maintenance in other systems. Their function in extraocular muscle is unknown. These studies suggest that this mouse model of nystagmus may allow molecular identification of candidate nystagmus-related genes.

TYR mutation in a Chinese population with oculocutaneous albinism: Molecular characteristics and ophthalmic manifestations

Oculocutaneous albinism (OCA) is a rare inherited disorder characterized by a partial or complete reduction of melanin biosynthesis that leads to hypopigmentation in the skin, hair and eyes. The OCA1 subtype is caused by mutations in TYR. The purpose of this study was to investigate the genetic and clinical ophthalmic characteristics of TYR mutations in patients with OCA. Herein, 51 probands with a clinical diagnosis of OCA were enrolled. Whole-exome sequencing and comprehensive ophthalmic examinations were performed. Overall, TYR mutations were detected in 37.3% (19/51) in the patients with OCA. Fifteen patients had compound heterozygous variants, and four cases had homozygous variants. Eleven different pathogenic variants in TYR were detected in these 19 patients, with missense, insertion, delins and nonsense in 71.1% (27/38), 15.8% (6/38), 2.6% (1/38), and 10.5% (4/38), respectively. Clinical examinations revealed that 84.2% (16/19) of patients were OCA1A, and 15.8% (3/19) were OCA1B. Most TYR probands (52.6%, 10/19) had moderate vision impairment, 15.8% (3/19) had severe visual impairment, 10.5% (2/19) exhibited blindness, only 5.3% (1/19) had mild visual impairment and 15.8% (3/19) were not available. Photophobia and nystagmus were found in 100% (19/19) of the patients. In addition, grade 4 foveal hypoplasia was detected in 100% (12/12) of the patients. In conclusion: The TYR patients exhibited severe ocular phenotypes: the majority (93.8%, 15/16) of them had a moderate vision impairment or worse, and 100% (12/12) had severe grade 4 foveal hypoplasia. These novel findings could provide insight into the understanding of OCA.

Free energy simulations to study mutational effect of a conserved residue, Trp24, on stability of human serum retinol-binding protein

Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a β-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the Trp→Leu, Trp→Phe, and Trp→Tyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24→Leu and Trp24→Phe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24→Tyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP. Communicated by Ramaswamy H. Sarma

Modulation of human transthyretin stability by the mutations at histidine 88 studied by free energy simulation.

Human transthyretin (TTR) is a homotetrameric plasma protein associated with a high percentage of β-sheet, which forms amyloid fibrils and accumulates in tissues or extracellular matrix to cause amyloid diseases. Free energy simulations based on all-atom molecular dynamics simulations were carried out to analyze the effects of the His88 → Arg, Phe, and Tyr mutations on the stability of human TTR. The calculated free energy change differences (ΔΔG) caused by the His → Arg, Phe, and Tyr mutations at position 88 are 6.48 ± 0.45, -9.99 ± 0.54, and 2.66 ± 0.33 kcal/mol, respectively. These calculated free energy change differences between wild type and the mutants are in excellent agreement with prior experimental values. Our simulation results show that the wild type of the TTR is more stable than H88R and H88Y mutants, whereas it is less stable than the H88F mutant. The free energy component analysis shows that the primary contribution to the free energy change difference (ΔΔG) for the His → Arg mutation arises from electrostatic interaction; the ΔΔG for the His → Phe mutation is from van der Waals and electrostatic interactions and that for the His → Tyr mutation from covalent interaction. The simulation results show that the free energy calculation with thermodynamic integration is beneficial for understanding the detailed microscopic mechanism of protein stability. The implications of the results for understanding stabilizing and destabilizing effect of the mutation and the contribution to protein stability are discussed.

Production of all male amelanotic red tilapia by combining MAS-GMT and tyrb mutation

Red tilapia (Oreochromis spp.), with male growing faster and larger than female, is becoming more and more popular for aquaculture in recent years. The culture of mono-sex red tilapia is of great value. However, the lacking of convenient and accurate sex-linked marker has limited the application of sex control in the breeding process of red tilapia. In addition, red tilapias show a wide range of color variation in each generation, which makes them not as valuable as pure red individuals. These are the bottlenecks of red tilapia culture. Tyrosinase is a key enzyme in melanin biosynthesis, and mutation of tyr is one of the main causes of albinism in vertebrates. In this study, we identified a sex-linked marker by sequencing and resequencing of Wang Lab dominant red tilapia (WLDRT, the red is controlled by single dominant gene) genome, selected YY supermales and established tyrb mutant line by CRISPR/Cas9 in red tilapia. The obtained homozygous XX and YY tyrb mutants displayed normal fertility and allowed continuous production of genetically male red tilapia (GMT) without blotches. Another tyrb mutant red tilapia line was established by crossing tyrb−/− Nile tilapia (NT) (Oreochromis niloticus) female with Wang Lab recessive red tilapia (WLRRT, the red is controlled by single recessive gene) male, followed by sibling crossing of the F1 heterozygous mutants. These mutants provide useful strains with improved commercial trait for red tilapia culture and good models for analyzing the genetic basis of body color formation in tilapia.

Simple embryo injection of long single-stranded donor templates with the CRISPR/Cas9 system leads to homology-directed repair in Xenopus tropicalis and Xenopus laevis.

We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).

Nonhuman Primate Model of Oculocutaneous Albinism with TYR and OCA2 Mutations.

Human visual acuity is anatomically determined by the retinal fovea. The ontogenetic development of the fovea can be seriously hindered by oculocutaneous albinism (OCA), which is characterized by a disorder of melanin synthesis. Although people of all ethnic backgrounds can be affected, no efficient treatments for OCA have been developed thus far, due partly to the lack of effective animal models. Rhesus macaques are genetically homologous to humans and, most importantly, exhibit structures of the macula and fovea that are similar to those of humans; thus, rhesus macaques present special advantages in the modeling and study of human macular and foveal diseases. In this study, we identified rhesus macaque models with clinical characteristics consistent with those of OCA patients according to observations of ocular behavior, fundus examination, and optical coherence tomography. Genomic sequencing revealed a biallelic p.L312I mutation in TYR and a homozygous p.S788L mutation in OCA2, both of which were further confirmed to affect melanin biosynthesis via in vitro assays. These rhesus macaque models of OCA will be useful animal resources for studying foveal development and for preclinical trials of new therapies for OCA.

Phospho-mutant activity assays provide evidence for alternative phospho-regulation pathways of the transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR.

Summary The key basic helix–loop–helix (bHLH) transcription factor in iron (Fe) uptake, FER‐LIKE IRON DEFICIENCY‐INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C‐terminus, a regulatory domain of FIT, provides a trigger for FIT activation.Here, we use phospho‐mutant activity assays and study phospho‐mimicking and phospho‐dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272.Phospho‐mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT‐activating partner bHLH039, promoter transactivation, and protein stability. Phospho‐mimicking Tyr mutations of FIT disturb fit mutant plant complementation.Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.

Mutational Analysis of TYR, OCA2, and SLC45A2 Genes in Chinese Families with Oculocutaneous Albinism.

BackgroundOculocutaneous albinism (OCA) is a group of heterogeneous autosomal recessive genetic disorder of melanin synthesis results in hypopigmented hair, skin, and eyes. OCA type 1, OCA type 2, and OCA type 4, which are respectively caused by mutations in TYR, OCA2, and SLC45A2 have high morbidity rates in Asia.Methods TYR, OCA2, and SLC45A2 mutation analysis was carried out on 18 nonconsanguineous OCA patients and four fetuses were included for prenatal diagnose. Three genes of all individuals were amplified by polymerase chain reaction and examined by Sanger sequencing. The pathogenicity of the detected mutations were analyzed by Mutation Taster, PolyPhen 2, and SIFT software, and the conservation of the substituted amino acids were analyzed by MEGA software.ResultsEleven TYR mutations, three OCA2 mutations, and two SLC45A2 mutations were identified in 14 OCA type 1 patients, two OCA type 2 patients, and two OCA type 4 patients. c.1021A>G, p.R341G in TYR, c.1096_1104del, p.V366*, and c.1079C>T, p.S360F in OCA2 were novel. One of the four fetuses carried compound heterozygous mutation of TYR and became spontaneous abortion, the other three carried no mutations and appeared normal at birth.ConclusionIn this study, specific clinical characteristics of OCA patients were described. Three novel pathogenic mutations were identified which will enrich the mutation spectrum of OCA, and the prenatal genetic screening in fetus at risk of OCA can provide vital information for genetic counseling.

Genetic analyses of oculocutaneous albinism types 1 and 2 with four novel mutations

BackgroundOculocutaneous albinism (OCA) is a human autosomal-recessive hypopigmentation disorder with hypopigmentation in the skin, hair, and eyes. OCA1 and OCA2 are caused by mutations of the TYR and OCA2 genes, respectively, which are responsible for most oculocutaneous albinism. However, the incidence of oculocutaneous albinism patients in Guangxi remains unclear.MethodsTo evaluate the molecular basis of oculocutaneous albinism in thirty-six patients in Guangxi, China. Peripheral venous blood samples were collected from these unrelated patients. The TYR and OCA2 genes of all individuals were analyzed by direct DNA sequencing and the sequences compared with are reference database and bioinformatics analysis.ResultsAmong the 36 OCA patients, 8(22.2%) were found mutations on TYR gene, 28 (77.8%) on OCA2. And we identified Twenty-seven different TYR and OCA2 mutations in these patients, including one novel TYR framshift mutation c.561_562insTTATTATGTGTCAAATTATCCCCCA, three novel OCA2 mutations: one nonsense mutation c.2195C > G(p.S732X), one deletation mutation(c.1139-1141delTGG), one missense mutations c.2495A > C(p.H832P). The population screening and the bioinformatic analysis to determined the effects of the mutations, which revealed these four novel mutations were pathogenic.ConclusionsThis study expands the mutation spectrum of oculocutaneous albinism. Four novel mutational alleles c.1139-1141delTGG, c.1832 T > C and c.2195C > G and of the OCA2 gene and c.561_562insTTATTATGTGTCAAATTATCCCCCA of TYR were associated with OCA. The genotype–phenotype correlations suggest that molecular diagnosis is more accurate and important in OCA.

TYR Gene in Llamas: Polymorphisms and Expression Study in Different Color Phenotypes.

Tyrosinase, encoded by TYR gene, is an enzyme that plays a major role in mammalian pigmentation. It catalyzes the oxidation of L-dihydroxy-phenylalanine (DOPA) to DOPA quinone, a precursor of both types of melanin: eumelanin and pheomelanin. TYR is commonly known as the albino locus since mutations in this gene result in albinism in several species. However, many other TYR mutations have been found to cause diluted phenotypes, like the Himalayan or chinchilla phenotypes in mice. The llama (Lama glama) presents a wide variety of coat colors ranging from non-diluted phenotypes (eumelanic and pheomelanic), through different degrees of dilution, to white. To investigate the possible contribution of TYR gene to coat color variation in llamas, we sequenced TYR exons and their flanking regions and genotyped animals with diluted, non-diluted, and white coat, including three blue-eyed white individuals. Moreover, we analyzed mRNA expression levels in skin biopsies by qPCR. TYR coding region presented nine SNPs, of which three were non-synonymous, c.428A > G, c.859G > T, and c.1490G > T. We also identified seven polymorphisms in non-coding regions, including two microsatellites, an homopolymeric repeat, and five SNPs: one in the promoter region (c.1-26C > T), two in the 3′-UTR, and two flanking the exons. Although no complete association was found between coat color and SNPs, c.1-26C > T was partially associated to diluted phenotypes. Additionally, the frequency of the G allele from c.428A > G was significantly higher in white compared to non-diluted. Results from qPCR showed that expression levels of TYR in white llamas were significantly lower (p < 0.05) than those in diluted and non-diluted phenotypes. Screening for variation in regulatory regions of TYR did not reveal polymorphisms that explain such differences. However, data from this study showed that TYR expression levels play a role in llama pigmentation.

Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system.

CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species.

Genetic studies of multiple consanguineous Pakistani families segregating oculocutaneous albinism identified novel and reported mutations.

Oculocutaneous albinism (OCA) is an autosomal-recessive disorder of a defective melanin pathway. The condition is characterized by hypopigmentation of hair, dermis, and ocular tissue. Genetic studies have reported seven nonsyndromic OCA genes, among which Pakistani OCA families mostly segregate TYR and OCA2 gene mutations. Here in the present study, we investigate the genetic factors of eight consanguineous OCA families from Pakistan. Genetic analysis was performed through single-nucleotide polymorphism (SNP) genotyping (for homozygosity mapping), whole exome sequencing (for mutation identification), Sanger sequencing (for validation and segregation analysis), and quantitative PCR (qPCR) (for copy number variant [CNV] validation). Genetic mapping in one family identified a novel homozygous deletion mutation of the entire TYRP1 gene, and a novel deletion of exon 19 in the OCA2 gene in two apparently unrelated families. In three further families, we identified homozygous mutations in TYR (NM_000372.4:c.1424G>A; p.Trp475*), NM_000372.4:c.895C>T; p.Arg299Cys), and SLC45A2 (NM_016180:c.1532C>T; p.Ala511Val). For the remaining two families, G and H, compound heterozygous TYR variants NM_000372.4:c.1037-7T>A, NM_000372.4:c.1255G>A (p.Gly419Arg), and NM_000372.4:c.1255G>A (p.Gly419Arg) and novel variant NM_000372.4:c.248T>G; (p.Val83Gly), respectively, were found. Our study further extends the evidence of TYR and OCA2 as genetic mutation hot spots in Pakistani families. Genetic screening of additional OCA cases may also contribute toward the development of Pakistani specific molecular diagnostic tests, genetic counseling, and personalized healthcare.

Characterization of a mutation in the zona pellucida module of Endoglin that causes Hereditary Hemorrhagic Telangiectasia

Hereditary hemorrhagic telangiectasia (HHT) is a vascular rare disease characterized by nose and gastrointestinal bleeding, skin and mucosa telangiectasias, and arteriovenous malformations in internal organs. HHT shows an autosomal dominant inheritance and a worldwide prevalence of approximately 1:5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG (HHT1) or ACVRL1 (HHT2) genes, which code for the membrane proteins Endoglin and Activin A Receptor Type II-Like Kinase 1 (ALK1), respectively, both belonging to the TGF-β/BMP signaling pathway. In this work, we describe a novel mutation in exon 9 of ENG (c.1145 G > A) found in five affected members of a family, all of them with characteristic symptoms of HHT. This mutation involves Cys382 residue of the Endoglin protein (p.Cys382 > Tyr) in the zona pellucida (ZP) module of its extracellular region. This is a critical residue involved in a conserved intrachain disulphide bond and in the correct folding of the protein. In fact, transfection studies in human cells using Endoglin expression vectors demonstrated that the p.Cys382 > Tyr mutation results in a marked reduction in the levels of the Endoglin protein. These results demonstrate the pathogenic role for this variant in HHT1 and confirm the key function of Cys382 in Endoglin expression.

The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering.

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.

Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase: insights from molecular dynamics simulations

Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine-uridine nucleoside hydrolase (CU-NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU-NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU-NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU-NH are identified. Regardless of the wild-type or mutant CU-NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU-NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU-NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.

Mild form of oculocutaneous albinism type 1: phenotypic analysis of compound heterozygous patients with the R402Q variant of the TYR gene

AimOculocutaneous albinism type 1 (OCA1) is due to TYR mutations. c.1205G>A/p.Arg402Gln (R402Q) is a thermosensitive variant of the TYR gene that has been reported to be responsible for mild forms...

Novel leptin receptor signaling mutants identify location and sex-dependent modulation of bone density, adiposity, and growth.

Leptin, a hormone primarily produced by adipocytes, contributes to the regulation of bone health by modulating bone density, growth and adiposity. Upon leptin binding, multiple sites of the long form of the leptin receptor (LepRb) are phosphorylated to trigger activation of downstream signaling pathways. To address the role of LepRb-signaling pathways in bone health, we compared the effects of three LepRb mutations on bone density, adiposity, and growth in male and female mice. The ∆65 mutation, which lacks the known tyrosine phosphorylation sites, caused obesity and the most dramatic bone phenotype marked by excessive bone adiposity, osteoporosis, and decreased growth, consistent with the phenotype of db/db and ob/ob mice that fully lack leptin receptor signaling. Mutation of LepRb Tyr 1138 , which results in an inability to recruit and phosphorylate signal transducer and activator of transcription 3, also caused obesity, but bone loss and adiposity were more dominant in male mice and no growth defect was observed. In contrast, mutation of LepRb Tyr 985 , which blocks SHP2/SOCS3 recruitment to LepRb and contributes to leptin hypersensitivity, promoted increased femur bone density only in male mice, while marrow adiposity and bone growth were not affected. Additional analyses of vertebral trabecular bone volume indicate that only the Tyr 1138 mutant mice exhibit bone loss in vertebrae. Together, our findings suggest that the phosphorylation status of specific sites of the LepRb contribute to the sex- and location-dependent bone responses to leptin. Unraveling the mechanisms by which leptin responses are sex- and location-dependent can contribute to the development of uniquely targeted osteoporosis therapies.