Conserved Amino Acid Residues Research Articles

Potential role of Methoprene-tolerant (Met) in methyl farnesoate-mediated vitellogenesis in the Chinese mitten crab (Eriocheir sinensis).

Methoprene-tolerant (Met) belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of nuclear transcriptional regulators and is a leading candidate receptor for juvenile hormone (JH III) in insects. Methyl farnesoate (MF) is a de-epoxide form of JH III that regulates many developmental processes in crustaceans, including reproduction, molting, and morphogenesis, much like JH III in insects. In this study, the full-length cDNA for Met was cloned from the Chinese mitten crab (Eriocheir sinensis) (EsMet). The amino acid sequence of EsMet contains three conserved domains (bHLH, PAS-A, and PASB) characteristic of the bHLH-PAS family, having six conserved amino acid residues specifically responsible for JH or MF binding. Tissue distribution analysis revealed that EsMet mRNA is highly expressed in the hepatopancreas. In addition, EsMet and EsVg expression in the hepatopancreas were found to be significantly increased in early endogenous vitellogenic oocytes (stage II) during ovarian development, and the hemolymph MF titer was significantly increased in late exogenous vitellogenic oocytes (stage III), indicating that EsMet is involved in vitellogenesis regulation. In vitro, MF addition markedly upregulated EsMet and EsVg expression in hepatopancreatic tissue, but only EsVg was induced in ovarian tissue. In vivo, EsMet and EsVg expression in the hepatopancreas were both significantly and synchronously increased after MF injection, but not in the ovaries. In addition, EsMet and EsVg expression were upregulated in the hepatopancreas after eyestalk ablation, while only EsVg expression was induced in the ovaries. Thus, our results indicate that Met may act as a receptor for MF in MF-mediated vitellogenesis in crustaceans.

Bi-Allelic Novel Variants in CLIC5 Identified in a Cameroonian Multiplex Family with Non-Syndromic Hearing Impairment

DNA samples from five members of a multiplex non-consanguineous Cameroonian family, segregating prelingual and progressive autosomal recessive non-syndromic sensorineural hearing impairment, underwent whole exome sequencing. We identified novel bi-allelic compound heterozygous pathogenic variants in CLIC5. The variants identified, i.e., the missense [NM_016929.5:c.224T>C; p.(L75P)] and the splicing (NM_016929.5:c.63+1G>A), were validated using Sanger sequencing in all seven available family members and co-segregated with hearing impairment (HI) in the three hearing impaired family members. The three affected individuals were compound heterozygous for both variants, and all unaffected individuals were heterozygous for one of the two variants. Both variants were absent from the genome aggregation database (gnomAD), the Single Nucleotide Polymorphism Database (dbSNP), and the UK10K and Greater Middle East (GME) databases, as well as from 122 apparently healthy controls from Cameroon. We also did not identify these pathogenic variants in 118 unrelated sporadic cases of non-syndromic hearing impairment (NSHI) from Cameroon. In silico analysis showed that the missense variant CLIC5-p.(L75P) substitutes a highly conserved amino acid residue (leucine), and is expected to alter the stability, the structure, and the function of the CLIC5 protein, while the splicing variant CLIC5-(c.63+1G>A) is predicted to disrupt a consensus donor splice site and alter the splicing of the pre-mRNA. This study is the second report, worldwide, to describe CLIC5 involvement in human hearing impairment, and thus confirms CLIC5 as a novel non-syndromic hearing impairment gene that should be included in targeted diagnostic gene panels.

Characterization of two sugar transporters responsible for efficient xylose uptake in an oleaginous yeast Candida tropicalis SY005

Microbial conversion of lignocellulosic feedstock to the target bioproduct requires efficient assimilation of its constituent sugars, a large part of which comprises of glucose and xylose. This study aims to identify and characterize sugar transporters capable of xylose uptake in an oleaginous strain of the industrially relevant yeast Candida tropicalis. In silico database mining resulted in two sugar transporter proteins- CtStp1 and CtStp2, containing conserved amino acid residues and motifs that have been previously reported to be involved in xylose transport in other organisms. Several softwares predicted the likelihood of 10–12 transmembrane (TM) helices to be present in both the Stps, while molecular modelling showed 12 TM helices that were organized into a typical structure found in the major facilitator superfamily of transporters. Docking with different sugars also predicted favorable interactions. Heterologous expression in a Saccharomyces cerevisiae strain harboring functional xylose metabolic genes validated the broad substrate specificity of the two Stps. Each transporter supported prominent growth of recombinant S. cerevisiae strains on six sugars including xylose at various concentrations. Expression of CtSTP1 and CtSTP2 along with the xylose metabolic genes in yeast transformants grown in presence of xylose was confirmed by transcript detection. Growth curve and sugar consumption profiles revealed uptake of both glucose and xylose simultaneously by the recombinant yeast strains, though CtStp1 showed relatively less effect of glucose repression in mixed sugars and was a better transporter of xylose than CtStp2. Such glucose-xylose utilizing efficient transporters can be effective tools for developing co-fermenting yeasts through genetic engineering in future, with noteworthy applications in renewable biomass utilization.

Comprehensive in-silico analysis of damage associated SNPs in hOCT1 affecting Imatinib response in chronic myeloid leukemia

Non-synonymous single nucleotide polymorphisms (nsSNPs) in hOCT1 (encoded by SLC22A1 gene) are expected to affect Imatinib uptake in chronic myeloid leukemia (CML). In this study, sequence homology-based genetic analysis of a set of 270 coding SNPs identified 18 nsSNPs to be putatively damaging/deleterious using eight different algorithms. Subsequently, based on conservation of amino acid residues, stability analysis, posttranscriptional modifications, and solvent accessibility analysis, the possible structural-functional relationship was established for high-confidence nsSNPs. Furthermore, based on the modeling results, some dissimilarities of mutant type amino acids from wild-type amino acids such as size, charge, interaction and hydrophobicity were revealed. Three highly deleterious mutations consisting of P283L, G401S and R402G in SLC22A1 gene that may alter the protein structure, function and stability were identified. These results provide a filtered data to explore the effect of uncharacterized nsSNP and find their association with Imatinib resistance in CML.

Characterization of a Membrane‐Bound O‐Acetyltransferase Involved in Trioxacarcin Biosynthesis Offers Insights into Its Catalytic Mechanism†

Summary of main observation and conclusionTrioxacarcin A (TXN‐A) is a polycyclic aromatic natural product with remarkable biological activity. TxnB11, a membrane‐bound O‐acetyltransferase involved in TXN‐A biosynthesis that is difficult for protein manipulation, is biochemically characterized here for its acetylation function on C4‐sugar. A proposed catalytic mechanism is supported by transmembrane helix analysis and site‐directed mutagenesis, in which four conserved amino acid residues His35, Ser71, His100 and His317 are essential for enzyme activity. We tested the substrate tolerance of TxnB11 to acyl donors in vitro and found that TxnB11 can also utilize propionyl‐CoA and glycolyl‐CoA, resulting in two new TXN analogs with anti‐tumor activity. This work provides a first understanding of the catalytic mechanism of this unusual acyltransferase family and presents good candidates for creating structural diversity for natural products.

Biological activities of a recombinant fortilin from Fenneropenaeus merguiensis.

Human Fortilin, an antiapoptotic protein, has also been implicated in several diseases; however, several potential uses of fortilin have also been proposed. Bearing the implications of fortilin in mind, fortilin analog, which has no complication with diseases, is required. Since a recombinant full-length fortilin from Fenneropenaeus merguiensis (rFm-Fortilin (FL)) reported only 44% (3e-27) homologous to human fortilin, therefore the biological activities of the Fm-Fortilin (FL) and its fragments (F2, F12, and F23) were investigated for potential use against HEMA toxicity from filling cement to pulp cell. The rFm-Fortilin FL, F2, 12, and F23 were expressed and assayed for proliferation activity. The rFm-Fortilin (FL) showed proliferation activity on human dental pulp cells (HDPCs) and protected the cells from 2-hydroxy-ethyl methacrylate (HEMA) at 1–20 ng/ml. In contrast, none of the rFm-Fortilin fragments promoted HDPC growth that may be due to a lack of three conserved amino acid residues together for binding with the surface of Rab GTPase for proliferative activity. In addition, rFm-Fortilin (FL) activated mineralization and trend to suppressed production of proinflammatory cytokines, including histamine (at 10 ng/ml) and TNF-α (at 100 ng/ml). Besides, the rFm-Fortilin (FL) did not mutate the Chinese hamster ovary (CHO) cell. Therefore, the rFm-Fortilin (FL) has the potential use as a supplementary medical material to promote cell proliferation in patients suffering severe tooth decay and other conditions.

Molecular characterization and pathogenicity of very virulent infectious bursal disease virus isolated from naturally infected turkey poults in Egypt.

This study was conducted to investigate the molecular characterization and pathogenicity of very virulent infectious bursal disease virus (vvIBDV) isolated from naturally infected turkey poults and possible spread to chickens. Thirty samples were collected from turkey poults in the vicinity or in the same backyards with chickens suspected to be infected with IBDV and from live bird markets from different localities in Dakahlia governorate, Egypt. There were no obvious clinical signs in tested turkey poults except dehydration and whitish diarrhoea in some birds with no mortality, and post-mortem lesions were observed in few birds as atrophied bursae, nephritis and petechial haemorrhages on thigh muscles. Reverse transcription polymerase chain reaction (RT-PCR), histopathological examination and immunohistochemistry were used for identification of the IBDV. Out of 30 tested samples, 17 samples (56.7%) were positive by RT-PCR. Phylogenetic analysis of VP2 gene of two selected IBDV strains (turkey 1 and turkey 2) showed a close genetic relationship to vvIBDV strains (serotype 1) isolated from chickens in Egypt and other countries with 93.1 to 95.99% identity for turkey 1 strain and 95.54 to 98.51% for turkey 2 strain. Both turkey 1 and turkey 2 strains were closely related to the Nigerian vvIBDV strain isolated from turkeys with 95.78% and 96.37% identity, respectively. Sequence analysis of both strains demonstrated that they have conserved amino acid residues of vvIBDV (I242, I294 and S299) and Y220F amino acid substitution which is very common in Egyptian vvIBDV chicken strains, while Turkey 1 strain has amino acid substitutions at A222P and I256V. Histopathological examination showed marked depletion of bursal lymphoid tissue. In conclusion, for the first time in Egypt, the molecular characterization and pathogenicity confirmed the presence of natural infection of turkey poults with vvIBDV (serotype 1) with possible spread to chickens causing severe economic losses.

Effect of Metal Ions on the Activity of Ten NAD-Dependent Formate Dehydrogenases.

NAD-dependent formate dehydrogenase (FDH) enzymes are frequently used in industrial and scientific applications. FDH is a reversible enzyme that reduces the NAD molecule to NADH and produces CO2 by oxidation of the formate ion, whereas it causes CO2 reduction in the reverse reaction. Some transition metal elements - Fe3+, Mo6+ and W6 + - can be found in the FDH structure of anaerobic and archaeal microorganisms, and these enzymes require cations and other redox-active cofactors for their FDH activity. While NAD-dependent FDHs do not necessarily require any metal cations, the presence of various metal cations can still affect FDH activities. To study the effect of 11 different metal ions, NAD-dependent FDH enzymes from ten different microorganisms were tested: Ancylobacter aquaticus (AaFDH), Candida boidinii (CboFDH), Candida methylica (CmFDH), Ceriporiopsis subvermispora (CsFDH), Chaetomium thermophilum (CtFDH), Moraxella sp. (MsFDH), Myceliophthora thermophila (MtFDH), Paracoccus sp. (PsFDH), Saccharomyces cerevisiae (ScFDH) and Thiobacillus sp. (TsFDH). It was found that metal ions (mainly Cu2+ and Zn2+) could have quite strong inhibition effects on several enzymes in the forward reaction, whereas several cations (Li+, Mg2+, Mn2+, Fe3+ and W6+) could increase the forward reaction of two FDHs. The highest activity increase (1.97 fold) was caused by Fe3+ in AaFDH. The effect on the reverse reaction was minimal. The modelled structures of ten FDHs showed that the active site is formed by 15 highly conserved amino acid residues spatially settling around the formate binding site in a conserved way. However, the residue differences at some of the sites close to the substrate do not explain the activity differences. The active site space is very tight, excluding water molecules, as observed in earlier studies. Structural examination indicated that smaller metal ions might be spaced close to the active site to affect the reaction. Metal ion size showed partial correlation to the effect on inhibition or activation. Affinity of the substrate may also affect the sensitivity to the metal's effect. In addition, amino acid differences on the protein surface may also be important for the metal ion effect.

Structural modelling of the lumenal domain of human GPAA1, the metallo-peptide synthetase subunit of the transamidase complex, reveals zinc-binding mode and two flaps surrounding the active site

BackgroundThe transamidase complex is a molecular machine in the endoplasmic reticulum of eukaryotes that attaches a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins after cleaving a C-terminal propeptide with a defined sequence signal. Its five subunits are very hydrophobic; thus, solubility, heterologous expression and complex reconstruction are difficult. Therefore, theoretical approaches are currently the main source of insight into details of 3D structure and of the catalytic process.ResultsIn this work, we generated model 3D structures of the lumenal domain of human GPAA1, the M28-type metallo-peptide-synthetase subunit of the transamidase, including zinc ion and model substrate positions. In comparative molecular dynamics (MD) simulations of M28-type structures and our GPAA1 models, we estimated the metal ion binding energies with evolutionary conserved amino acid residues in the catalytic cleft. We find that canonical zinc binding sites 2 and 3 are strongest binders for Zn1 and, where a second zinc is available, sites 2 and 4 for Zn2. Zinc interaction of site 5 with Zn1 enhances upon substrate binding in structures with only one zinc. Whereas a previously studied glutaminyl cyclase structure, the best known homologue to GPAA1, binds only one zinc ion at the catalytic site, GPAA1 can sterically accommodate two. The M28-type metallopeptidases segregate into two independent branches with regard to one/two zinc ion binding modality in a phylogenetic tree where the GPAA1 family is closer to the joint origin of both groups. For GPAA1 models, MD studies revealed two large loops (flaps) surrounding the active site being involved in an anti-correlated, breathing-like dynamics.ConclusionsIn the light of combined sequence-analytic and phylogenetic arguments as well as 3D structural modelling results, GPAA1 is most likely a single zinc ion metallopeptidase. Two large flaps environ the catalytic site restricting access to large substrates.ReviewersThis article was reviewed by Thomas Dandekar (MD) and Michael Gromiha.

Conserved Amino Acid Residues that Affect Structural Stability of Candida boidinii Formate Dehydrogenase.

The NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme's thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73°C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25°C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies.

Novel compound heterozygous FBXO7 mutations in a family with early onset Parkinson's disease

BackgroundMutations in the F-box protein 7 (FBXO7) gene result in autosomal recessive parkinsonism. This usually manifests as early-onset parkinsonian-pyramidal syndrome but patients exhibit high phenotypic variability. Here we describe the findings of a Yemeni family with two novel FBXO7 mutations. MethodsClinical data and DNA were available for three siblings with early-onset parkinsonism together with their parents and three unaffected siblings. A targeted next generation sequencing panel was used to screen the proband for mutations in 14 genes known to cause a parkinsonian disorder. In addition, SNCA, PARK2, PINK1, and PARK7 were screened for copy number variants. ResultsThe proband carried two novel compound heterozygous FBXO7 mutations: a missense mutation in exon 1 (p.G39R; c.115G > A) and a frameshift mutation in exon 5 (p.L280fs; c.838del). The mutations segregated with disease in the family with the exception of a potentially pre-symptomatic individual whose age was below the age of onset in two of their three affected siblings. P.G39R occurred at a highly conserved amino acid residue and both mutations were predicted to be deleterious in silico. In contrast to most reported families, the phenotype in this pedigree was consistent with clinically typical Parkinson's disease (PD) with a lack of pyramidal signs and good response to dopaminergic therapy. ConclusionsOur study expands the phenotype associated with FBXO7 to include early-onset PD and broadens the list of causative mutations. These data suggest that FBXO7 should be included in clinical genetic testing panels for PD, particularly in patients with early onset or a recessive inheritance pattern.

Expression, Affinity, and Functional Characterization of the Specific Binding of Two Putative Pheromone-Binding Proteins in the Omnivorous German Cockroach Blattella germanica.

The German cockroach Blattella germanica (L.) is an important pest in medical, veterinary, and public health. Studies on the olfaction mechanism of hemimetabolous insects have rarely been reported, especially in cockroaches. Pheromone-binding proteins (PBPs) play a vital role in insect sex pheromone recognition, which solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors. In this study, two potential PBPs (BgerOBP26 and BgerOBP40) were identified on the basis of their biased expression in male antennae using tissue transcriptome data and verified by the quantitative real-time polymerase chain reaction approach. We then expressed and purified the two identified odorant-binding proteins (OBPs) using the Escherichia coli expression system and affinity purification. In vitro binding studies showed that the two OBPs display stronger binding affinities to the female volatile sex pheromone blattellaquinone than to its analogues and contact sex pheromone components. Finally, three-dimensional modeling of the two OBPs and dock conformation with sex pheromone molecules showed BgerOBP26 has a larger odorant cavity and more conservative active amino acid residues than BgerOBP40. These results illuminated the binding characteristics of potential PBPs of B. germanica, which could lay the groundwork for improved understanding of many aspects of the chemical ecology of B. germanica. Moreover, this information complements the understanding of the olfactory molecular mechanism in cockroaches and provides potential gene targets for B. germanica control.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Characterization and Map-Based Cloning of the Novel Rice Yellow Leaf Mutant yl3

Chlorophyll (Chl) is an essential molecule for harvesting light energy in photosynthetic organisms. We isolated yellow leaf 3 (yl3), a rice Chl-deficient mutant, which showed yellow leaves with decreased Chl content and abnormal chloroplast development. Positional cloning revealed that YL3 encodes OsCHLH, a subunit of magnesium-chelatase. In yl3, one amino acid substitution occurs at the C-terminal region of a highly conserved amino acid residue. YL3 is constitutively expressed in all tissues. Compared with the wild type, yl3 was found to be sensitive to low temperature. The expression levels of genes for Chl biosynthesis, photosynthesis and chloroplast development were strongly reduced in yl3. These results provide novel insights regarding how rice chloroplast development and Chl synthesis are regulated by YL3.

In Silico Finding of Key Interaction Mediated α3β4 and α7 Nicotinic Acetylcholine Receptor Ligand Selectivity of Quinuclidine-Triazole Chemotype.

The selective binding of six (S)-quinuclidine-triazoles and their (R)-enantiomers to nicotinic acetylcholine receptor (nAChR) subtypes α3β4 and α7, respectively, were analyzed by in silico docking to provide the insight into the molecular basis for the observed stereospecific subtype discrimination. Homology modeling followed by molecular docking and molecular dynamics (MD) simulations revealed that unique amino acid residues in the complementary subunits of the nAChR subtypes are involved in subtype-specific selectivity profiles. In the complementary β4-subunit of the α3β4 nAChR binding pocket, non-conserved AspB173 through a salt bridge was found to be the key determinant for the α3β4 selectivity of the quinuclidine-triazole chemotype, explaining the 47–327-fold affinity of the (S)-enantiomers as compared to their (R)-enantiomer counterparts. Regarding the α7 nAChR subtype, the amino acids promoting a however significantly lower preference for the (R)-enantiomers were the conserved TyrA93, TrpA149 and TrpB55 residues. The non-conserved amino acid residue in the complementary subunit of nAChR subtypes appeared to play a significant role for the nAChR subtype-selective binding, particularly at the heteropentameric subtype, whereas the conserved amino acid residues in both principal and complementary subunits are essential for ligand potency and efficacy.

Mechanism and potential sites of potassium interaction with glutamate transporters.

In the mammalian glutamate transporters, countertransported intracellular K+ is essential for relocating the glutamate binding site to the extracellular side of the membrane. This K+-dependent process is believed to be rate limiting for the transport cycle. In contrast, extracellular K+ induces glutamate release upon transporter reversal. Here, we analyzed potential K+ binding sites using molecular dynamics (MD) simulations and site-directed mutagenesis. Two candidate sites were identified by spontaneous K+ binding in MD simulations, one site (K1 site) overlapping with the Na1 Na+ binding site and the K2 site being localized under hairpin loop 2 (HP2). Mutations to conserved amino acid residues in these sites resulted in several transporters that were defective in K+-induced reverse transport and which bound K+ with reduced apparent affinity compared with the wild-type transporter. However, external K+ interaction was abolished in only one mutant transporter EAAC1D454A in the K1 site. Our results, for the first time, directly demonstrate effects of K1-site mutations on K+ binding, in contrast to previous reports on K+ binding sites based on indirect evidence. We propose that K+ binding to the K1 site is responsible for catalyzing the relocation step, whereas binding to the K2 site may have an as-of-yet unidentified regulatory function.

Highly conserved amino acid residues in apolipoprotein A1 discordantly induce high density lipoprotein assembly in vitro and in vivo

ObjectiveApolipoprotein A1 (APOA1) is essential to reverse cholesterol transport, a physiologically important process that protects against atherosclerotic cardiovascular disease. APOA1 is a 28 kDa protein comprising multiple lipid-binding amphiphatic helices initialized by proline residues, which are conserved across multiple species. We tested the hypothesis that the evolutionarily conserved residues are essential to high density lipoprotein (HDL) function. ApproachWe used biophysical and physiological assays of the function of APOA1P➔A variants, i.e., rHDL formation via dimyristoylphosphatidylcholine (DMPC) microsolubilization, activation of lecithin: cholesterol acyltransferase, cholesterol efflux from human monocyte-derived macrophages (THP-1) to each variant, and comparison of the size and composition of HDL from APOA1−/− mice receiving adeno-associated virus delivery of each human variant. ResultsDifferences in microsolubilization were profound and showed that conserved prolines, especially those in the C-terminus of APOA1, are essential to efficient rHDL formation. In contrast, P➔A substitutions produced small changes (−25 to +25%) in rates of cholesterol efflux and no differences in the rates of LCAT activation. The HDL particles formed following ectopic expression of each variant in APOA1−/− mice were smaller and more heterogeneous than those from control animals. ConclusionStudies of DMPC microsolubilization show that proline residues are essential to the optimal interaction of APOA1 with membranes, the initial step in cholesterol efflux and HDL production. In contrast, P➔A substitutions modestly reduce the cholesterol efflux capacity of APOA1, have no effect on LCAT activation, but according to the profound reduction in the size of HDL formed in vivo, P➔A substitutions alter HDL biogenesis, thereby implicating other cellular and in vivo processes as determinants of HDL metabolism and function.

A novel TFG c.793C>G mutation in a Chinese pedigree with Charcot‐Marie‐Tooth disease 2

IntroductionMutations within TFG gene were recently reported to cause Charcot‐Marie‐Tooth disease 2 (CMT2). However, only few pedigrees were documented so far. Here, we reported a Chinese CMT2 pedigree with 8 affected cases and a novel TFG mutation.MethodsClinical evaluation and electrophysiological study were performed in all the affected individuals. Whole‐exome sequencing was conducted, followed by the Sanger sequencing and co‐segregation analysis to verify the variants.ResultsAll cases presented with a phenotype of CMT2, including slowly progressive symmetrical muscle atrophy and weakness predominantly in the distal limbs. Sensory loss in the distal limbs was present in the proband and his father. Age at onset ranged from 37 to 44 years, and was younger in male cases, compared with female cases. Nerve conduction study revealed normal motor nerve conduction velocity but decreased compound muscle action potential. Electromyography test revealed fibrillation potential and positive sharp waves. The creatine kinase activity was increased in all cases. After genetic investigations, we identified a novel TFG c.793C>G (p.Pro265Ala) mutation in the family. This mutation alters the conserved amino acid residue and is absent in 1000G, ExAC, dbSNP, EP6500, and 200 in‐house controls. It co‐segregated with the disease in the family.ConclusionsOur report provided additional evidence that the heterozygous TFG mutations were associated with CMT2.

Bi-allelic Mutations in ALDH5A1 is associated with succinic semialdehyde dehydrogenase deficiency and severe intellectual disability

Homozygous mutations of ALDH5A1 have been reportedly associated with Succinic semialdehyde dehydrogenase deficiency (SSADHD) that affects gamma-aminobutyric acid (GABA) catabolism and evinces a wide range of clinical phenotype from mild intellectual disability to severe neurodegenerative disorders. We report clinical and molecular data of a Lor family with 2 affected members presenting with severe intellectual disability, developmental delay, and generalized tonic-clonic seizures. A comprehensive genetic study that included whole-exome sequencing identified a homozygous missense substitution (NM_001080:c.G1321A:p.G441R) in ALDH5A1 (Aldehyde Dehydrogenase 5 Family Member A1) gene, consistent with clinical phenotype in the patients and co-segregating with the disease in the family. The non-synonymous mutation, p.G441R, affects a highly conserved amino acid residue, which is expected to cause a severe destabilization of the enzyme. Protein modeling demonstrated an impairment of the succinic semialdehyde (SSA) binding tunnel accessibility, and the anticipation of the protein folding stability and dynamics was a decrease in the free energy by 4.02 kcal/mol. Consistent with these in silico findings, excessive γ -hydroxybutyrate (GHB) could be detected in patients’ urine as the byproduct of the GABA pathway.SSADHD, Succinic semialdehyde dehydrogenase deficiency; GABA, gamma-aminobutyric acid; ALDH5A1, Aldehyde Dehydrogenase 5 Family Member A1; GHB, γ –hydroxybutyrate; SSA, succinic semi aldehyde; WISC, Wechsler Intelligence Scale for Children; CNS, central nervous system ; EEG, electroencephalography; EEEF, empirical effective energy functions; ASD, autism spectrum disorder; ADHD, attention deficit hyperactivity disorder; IQ, intelligence quotient; EMG, electromyography; NCV, nerve conduction velocity; CP, cerebral palsy

Dietary flavonoid fisetin binds human SUMO1 and blocks sumoylation of p53.

Flavonoids are plant-derived compounds that occur abundantly in fruits and vegetables and have been shown to possess potent anti-cancer, antioxidant, and anti-inflammatory properties. However, their direct targets and molecular mechanism of action are not well characterized, hampering exploitation of the beneficial properties of flavonoids for drug development. Small ubiquitin-related modifier 1 (SUMO1) is attached to target proteins as part of a post-translational modification system implicated in a myriad of cellular processes from nuclear trafficking to transcriptional regulation. Using a combination of surface plasmon resonance, differential scanning fluorimetry and fluorescence quenching studies, we provide evidence for direct binding of the dietary flavonoid fisetin to human SUMO1. Our NMR chemical shift perturbation analyses reveal that binding to fisetin involves four conserved amino acid residues (L65, F66, E67, M82) previously shown to be important for conjugation of SUMO1 to target proteins. In vitro sumoylation experiments indicate that fisetin blocks sumoylation of tumor suppressor p53, consistent with fisetin negatively affecting post-translational modification and thus the biological activity of p53. A series of differential scanning fluorimetry experiments suggest that high concentrations of fisetin result in destabilization and unfolding of SUMO1, presenting a molecular mechanism by which flavonoid binding affects its activity. Overall, our data establish a novel direct interaction between fisetin and SUMO1, providing a mechanistic explanation for the ability of fisetin to modulate multiple key signaling pathways inside cells.