Lnc-HIBADH-4 Regulates Autophagy-Lysosome Pathway in Amyotrophic Lateral Sclerosis by Targeting Cathepsin D.

Amyotrophic lateral sclerosis (ALS) is the most prevalent and lethal class of severe motor neuron diseases (MND) with no efficacious treatment. The pathogenic mechanisms underlying ALS remain unclear. Nearly 90% of patients exhibit sporadic onset (sALS). Therefore, elucidating the pathophysiology of ALS is imperative. Long non-coding RNA (lncRNA) is a large class of non-coding RNAs that regulate transcription, translation, and post-translational processes. LncRNAs contribute to the pathogenesis of diverse neurodegenerative disorders and hold promise as targets for interference in the realm of neurodegeneration. However, the mechanisms of which lncRNAs are involved in ALS have not been thoroughly investigated. We identified and validated a downregulated lncRNA, lnc-HIBADH-4, in ALS which correlated with disease severity and overall survival. Lnc-HIBADH-4 acted as a "molecular sponge" regulating lysosomal function through the lnc-HIBADH-4/miR-326/CTSD pathway, thereby impacting autophagy-lysosome dynamics and the levels of cell proliferation and apoptosis. Therefore, this study discovered and revealed the role of lnc-HIBADH-4 in the pathogenesis of ALS. With further research, lnc-HIBADH-4 is expected to provide a new biomarker in the diagnosis and treatment of ALS.

A Genetic Polymorphism in the WDR72 Gene is Associated With Calcium Nephrolithiasis in the Chinese Han Population.

A previous genome-wide association study (GWAS) reported several novel loci for nephrolithiasis in British and Japanese population, some of which were predicted to influence CaSR signaling. In this study, we aimed to evaluate the association of these loci with calcium nephrolithiasis in Chinese Han population. We performed a case-control association analysis involving 691 patients with calcium nephrolithiasis and 1008 control subjects. We were able to genotype a total of 17 single-nucleotide polymorphisms (SNPs), which were previously reported to be significantly associated with nephrolithiasis in GWAS. rs578595 at WDR72 was significantly associated with calcium nephrolithiasis in Chinese Han population (p < 0.001, OR = 0.617). Moreover, rs12654812 at SLC34A1 (p = 0.0427, OR = 1.170), rs12539707 at HIBADH (p = 0.0179, OR = 0.734), rs1037271 at DGKH (p = 0.0096, OR = 0.828) and rs12626330 at CLDN14 (p = 0.0080, OR = 1.213) indicated suggestive associations with calcium nephrolithiasis. Our results elucidated the significance of genetic variation at WDR72, DGKH, CLDN14, SLC34A1, and HIBADH in Chinese patients with nephrolithiasis. Since polymorphisms of WDR72, DGKH, and CLDN14 are predicted to influence in CaSR signaling, our results emphasized the role of abnormal calcium homeostasis in calcium nephrolithiasis.

3-Hydroxyisobutyric acid dehydrogenase deficiency: Expanding the clinical spectrum and quantitation of D- and L-3-Hydroxyisobutyric acid by an LC-MS/MS method.

A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients.

3-Hydroxyisobutyrate dehydrogenase (HIBADH) deficiency-A novel disorder of valine metabolism.

3-Hydroxyisobutyric acid (3HiB) is an intermediate in the degradation of the branched-chain amino acid valine. Disorders in valine degradation can lead to 3HiB accumulation and its excretion in the urine. This article describes the first two patients with a new metabolic disorder, 3-hydroxyisobutyrate dehydrogenase (HIBADH) deficiency, its phenotype and its treatment with a low-valine diet. The detected mutation in the HIBADH gene leads to nonsense-mediated mRNA decay of the mutant allele and to a complete loss-of-function of the enzyme. Under strict adherence to a low-valine diet a rapid decrease of 3HiB excretion in the urine was observed. Due to limited patient numbers and intrafamilial differences in phenotype with one affected and one unaffected individual, the clinical phenotype of HIBADH deficiency needs further evaluation.

Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis.

Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.

Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae.

Biomass, the most abundant carbon source on the planet, may in the future become the primary feedstock for production of fuels and chemicals, replacing fossil feedstocks. This will, however, require development of cell factories that can convert both C6 and C5 sugars present in lignocellulosic biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP), a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or ß-alanine intermediates into a xylose-consuming yeast. Using controlled fed-batch cultivation, we obtained 7.37±0.17 g 3HP L-1 in 120 hours with an overall yield of 29±1% Cmol 3HP Cmol-1 xylose. This study is the first demonstration of the potential of using S. cerevisiae for production of 3HP from the biomass sugar xylose.

Identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants.

NADPH-dependent glyoxylate reductases from Arabidopsis thaliana (AtGLYR) convert both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. The primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the ß-HAD (ß-hydroxyacid dehydrogenase) protein family, whose members include 3-hydroxyisobutyrate dehydrogenase, tartronate semialdehyde reductase and 6-phosphogluconate dehydrogenase. Here, site-directed mutagenesis was utilized to identify catalytically important amino acid residues for glyoxylate reduction in AtGLYR1. Kinetic studies and binding assays established that Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis. The low activity of the mutant enzymes precluded kinetic studies with succinic semialdehyde. The crystal structure of AtGLYR1 in the absence of substrate was solved to 2.1Å by molecular replacement using a previously unrecognized member of the ß-HAD family, cytokine-like nuclear factor, thereby enabling the 3-D structure of the protein to be modeled with substrate and co-factor. Structural alignment of AtGLYR1 with ß-HAD family members provided support for the essentiality of Lys170, Phe173, Asp239, Ser121, Asn174 and Thr95 in the active site and preliminary support for an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule. This information established that AtGLYR1 is a member of the ß-HAD protein family. Sequence and activity comparisons indicated that AtGLYR1 and the plastidial AtGLYR2 possess structural features that are absent in Arabidopsis hydroxypyruvate reductases and probably account for their stronger preference for glyoxylate over hydroxypyruvate.