34 research outputs found
Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy
Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function
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How lipid droplets "TAG" along: Glycerolipid synthetic enzymes and lipid storage.
Triacylglycerols (TAG) serve as the predominant form of energy storage in mammalian cells, and TAG synthesis influences conditions such as obesity, fatty liver, and insulin resistance. In most tissues, the glycerol 3-phosphate pathway enzymes are responsible for TAG synthesis, and the regulation and function of these enzymes is therefore important for metabolic homeostasis. Here we review the sites and regulation of glycerol-3-phosphate acyltransferase (GPAT), acylglycerol-3-phosphate acyltransferase (AGPAT), lipin phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT) enzyme action. We highlight the critical roles that these enzymes play in human health by reviewing Mendelian disorders that result from mutation in the corresponding genes. We also summarize the valuable insights that genetically engineered mouse models have provided into the cellular and physiological roles of GPATs, AGPATs, lipins and DGATs. Finally, we comment on the status and feasibility of therapeutic approaches to metabolic disease that target enzymes of the glycerol 3-phosphate pathway. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink
Identification and biochemical characterization of an acid sphingomyelinase-like protein from the bacterial plant pathogen Ralstonia solanacearum that hydrolyzes ATP to AMP but not sphingomyelin to ceramide.
Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases
List of bacterial aSMase homologues identified by a BLAST search.
<p>List of bacterial aSMase homologues identified by a BLAST search.</p
Michaelis-Menten values for pNP-based substrates.
<p>Michaelis-Menten values for pNP-based substrates.</p
Domain architecture and sequence alignment of RsASML and human aSMase.
<p>(a) Domain architecture of acid SMase and acid-SMase-like proteins. Human aSMase contains three domains: a SAP (<u>S</u>phingolipid-<u>A</u>ctivating <u>P</u>rotein) involved in lipid binding, a <u>M</u>etallo<u>P</u>hos<u>P</u>hatase (MPP) catalytic domain, and a C-terminal domain required for activity but of unknown function. RsASML, from the bacteria <i>R. solanacearum</i>, and two human proteins, acid SMase-like 3a (aSML3a) and aSML3b of unknown function, share homology with the catalytic and C-terminal domains of human aSMase. (b) Sequence alignment of human aSMase and RsASML highlighting predicted secondary structure elements using Jpred3 (black = beta strands, grey = alpha helices). Black circles above denote identical residues. Asterisks below sequence indicate conserved residues identified in Niemann-Pick Type A or B patients. A known disulfide cysteine pair in human aSMase, conserved in RsASML, is noted.</p
RsASML catalyzes the hydrolysis of ATP and ADP to AMP.
<p>HPLC chromatograms of adenosine-based nucleotides incubated with reaction buffer or RsASML protein. All reactions were carried out in 10 mM HEPES, pH 8.0, 1 mM NiCl<sub>2</sub> with 1 µM RsASML protein.</p
Michaelis-Menten kinetics of RsASML vs. different pNP-based substrates.
<p>Concentration dependence of RsASML activity towards (a) pNPP, (b) pNPPC, (c) pNP-TMP. All reactions were carried out in 10 mM HEPES, pH 8.0, 1 mM NiCl<sub>2</sub>, with 100 nM RsASML protein.</p
K<sub>metal</sub> and V<sub>max</sub> values for NiCl<sub>2</sub> and MnCl<sub>2</sub>.
<p>K<sub>metal</sub> and V<sub>max</sub> values for NiCl<sub>2</sub> and MnCl<sub>2</sub>.</p