Publications by authors named "Vladimir Saudek"

33 Publications

Phenotypic characterization of Adig null mice suggests roles for adipogenin in the regulation of fat mass accrual and leptin secretion.

Cell Rep 2021 Mar;34(10):108810

Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, Cambridgeshire CB2 0QQ, UK. Electronic address:

Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion.
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http://dx.doi.org/10.1016/j.celrep.2021.108810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966854PMC
March 2021

GDF15: A Hormone Conveying Somatic Distress to the Brain.

Endocr Rev 2020 08;41(4)

MRC Metabolic Diseases Unit, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK.

GDF15 has recently gained scientific and translational prominence with the discovery that its receptor is a GFRAL-RET heterodimer of which GFRAL is expressed solely in the hindbrain. Activation of this receptor results in reduced food intake and loss of body weight and is perceived and recalled by animals as aversive. This information encourages a revised interpretation of the large body of previous research on the protein. GDF15 can be secreted by a wide variety of cell types in response to a broad range of stressors. We propose that central sensing of GDF15 via GFRAL-RET activation results in behaviors that facilitate the reduction of exposure to a noxious stimulus. The human trophoblast appears to have hijacked this signal, producing large amounts of GDF15 from early pregnancy. We speculate that this encourages avoidance of potential teratogens in pregnancy. Circulating GDF15 levels are elevated in a range of human disease states, including various forms of cachexia, and GDF15-GFRAL antagonism is emerging as a therapeutic strategy for anorexia/cachexia syndromes. Metformin elevates circulating GDF15 chronically in humans and the weight loss caused by this drug appears to be dependent on the rise in GDF15. This supports the concept that chronic activation of the GDF15-GFRAL axis has efficacy as an antiobesity agent. In this review, we examine the science of GDF15 since its identification in 1997 with our interpretation of this body of work now being assisted by a clear understanding of its highly selective central site of action.
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http://dx.doi.org/10.1210/endrev/bnaa007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299427PMC
August 2020

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Mol Biol Cell 2019 03 16;30(5):703-716. Epub 2019 Jan 16.

Laboratoire de Physique de l'Ecole Normale Supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université Paris-Diderot, Sorbonne Paris Cité, Paris, France.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a "hierarchical" manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1-3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both "naked" and phospholipid-coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1-3 to LDs.
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http://dx.doi.org/10.1091/mbc.E18-08-0534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589688PMC
March 2019

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.

Dev Cell 2018 05 10;45(4):481-495.e8. Epub 2018 May 10.

Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK. Electronic address:

Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.
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http://dx.doi.org/10.1016/j.devcel.2018.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971203PMC
May 2018

The metabolic syndrome- associated small G protein ARL15 plays a role in adipocyte differentiation and adiponectin secretion.

Sci Rep 2017 12 14;7(1):17593. Epub 2017 Dec 14.

The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK.

Common genetic variants at the ARL15 locus are associated with plasma adiponectin, insulin and HDL cholesterol concentrations, obesity, and coronary atherosclerosis. The ARL15 gene encodes a small GTP-binding protein whose function is currently unknown. In this study adipocyte-autonomous roles for ARL15 were investigated using conditional knockdown of Arl15 in murine 3T3-L1 (pre)adipocytes. Arl15 knockdown in differentiated adipocytes impaired adiponectin secretion but not adipsin secretion or insulin action, while in preadipocytes it impaired adipogenesis. In differentiated adipocytes GFP-tagged ARL15 localized predominantly to the Golgi with lower levels detected at the plasma membrane and intracellular vesicles, suggesting involvement in intracellular trafficking. Sequencing of ARL15 in 375 severely insulin resistant patients identified four rare heterozygous variants, including an early nonsense mutation in a proband with femorogluteal lipodystrophy and non classical congenital adrenal hyperplasia, and an essential splice site mutation in a proband with partial lipodystrophy and a history of childhood yolk sac tumour. No nonsense or essential splice site mutations were found in 2,479 controls, while five such variants were found in the ExAC database. These findings provide evidence that ARL15 plays a role in adipocyte differentiation and adiponectin secretion, and raise the possibility that human ARL15 haploinsufficiency predisposes to lipodystrophy.
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http://dx.doi.org/10.1038/s41598-017-17746-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730586PMC
December 2017

Potential dual function of PQ-loop proteins such as cystinosin.

Authors:
Vladimir Saudek

J Biol Chem 2017 09;292(36):15133

From the Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge Metabolic Research Laboratories, CB2 OQQ Cambridge, United Kingdom

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http://dx.doi.org/10.1074/jbc.L117.795278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592687PMC
September 2017

Obesity-associated gene has a role in the central control of appetite and body weight regulation.

Proc Natl Acad Sci U S A 2017 08 15;114(35):9421-9426. Epub 2017 Aug 15.

University of Cambridge Metabolic Research Laboratories, Level 4, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom;

An intergenic region of human chromosome 2 (2p25.3) harbors genetic variants which are among those most strongly and reproducibly associated with obesity. The gene closest to these variants is , although the molecular mechanisms mediating these effects remain entirely unknown. expression in the murine hypothalamic paraventricular nucleus (PVN) was altered by changes in nutritional state. Germline loss of in mice resulted in increased body weight, which was exacerbated by high fat diet and driven by increased food intake. Selective overexpression of in the PVN of wild-type mice reduced food intake and also increased energy expenditure. We provide evidence that TMEM18 has four, not three, transmembrane domains and that it physically interacts with key components of the nuclear pore complex. Our data support the hypothesis that itself, acting within the central nervous system, is a plausible mediator of the impact of adjacent genetic variation on human adiposity.
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http://dx.doi.org/10.1073/pnas.1707310114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584443PMC
August 2017

The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of development.

PeerJ 2017 6;5:e3390. Epub 2017 Jun 6.

Biocev, First Faculty of Medicine, Charles University, Prague, Czech Republic.

The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.
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http://dx.doi.org/10.7717/peerj.3390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464003PMC
June 2017

Human biallelic MFN2 mutations induce mitochondrial dysfunction, upper body adipose hyperplasia, and suppression of leptin expression.

Elife 2017 04 19;6. Epub 2017 Apr 19.

The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, United Kingdom.

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies.
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http://dx.doi.org/10.7554/eLife.23813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422073PMC
April 2017

FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.

Nat Struct Mol Biol 2017 01 5;24(1):23-29. Epub 2016 Dec 5.

Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD hamster cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation-defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.
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http://dx.doi.org/10.1038/nsmb.3337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221731PMC
January 2017

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1-3.

J Biol Chem 2016 Mar 7;291(13):6664-78. Epub 2016 Jan 7.

From the University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom,

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs inSaccharomyces cerevisiae,demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targetingin vivoandin vitro Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.
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http://dx.doi.org/10.1074/jbc.M115.691048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807253PMC
March 2016

Perilipin-related protein regulates lipid metabolism in C. elegans.

PeerJ 2015 1;3:e1213. Epub 2015 Sep 1.

Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague , Albertov, Prague , Czech Republic.

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.
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http://dx.doi.org/10.7717/peerj.1213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562238PMC
September 2015

FTO is necessary for the induction of leptin resistance by high-fat feeding.

Mol Metab 2015 Apr 7;4(4):287-98. Epub 2015 Feb 7.

University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK.

Objective: Loss of function FTO mutations significantly impact body composition in humans and mice, with Fto-deficient mice reported to resist the development of obesity in response to a high-fat diet (HFD). We aimed to further explore the interactions between FTO and HFD and determine if FTO can influence the adverse metabolic consequence of HFD.

Methods: We studied mice deficient in FTO in two well validated models of leptin resistance (HFD feeding and central palmitate injection) to determine how Fto genotype may influence the action of leptin. Using transcriptomic analysis of hypothalamic tissue to identify relevant pathways affected by the loss of Fto, we combined data from co-immunoprecipitation, yeast 2-hybrid and luciferase reporter assays to identify mechanisms through which FTO can influence the development of leptin resistant states.

Results: Mice deficient in Fto significantly increased their fat mass in response to HFD. Fto (+/-) and Fto (-/-) mice remained sensitive to the anorexigenic effects of leptin, both after exposure to a HFD or after acute central application of palmitate. Genes encoding components of the NFкB signalling pathway were down-regulated in the hypothalami of Fto-deficient mice following a HFD. When this pathway was reactivated in Fto-deficient mice with a single low central dose of TNFα, the mice became less sensitive to the effect of leptin. We identified a transcriptional coactivator of NFкB, TRIP4, as a binding partner of FTO and a molecule that is required for TRIP4 dependent transactivation of NFкB.

Conclusions: Our study demonstrates that, independent of body weight, Fto influences the metabolic outcomes of a HFD through alteration of hypothalamic NFкB signalling. This supports the notion that pharmacological modulation of FTO activity might have the potential for therapeutic benefit in improving leptin sensitivity, in a manner that is influenced by the nutritional environment.
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http://dx.doi.org/10.1016/j.molmet.2015.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354923PMC
April 2015

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance.

J Clin Invest 2014 Sep 8;124(9):4028-38. Epub 2014 Aug 8.

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.
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http://dx.doi.org/10.1172/JCI73264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151221PMC
September 2014

Perilipins 2 and 3 lack a carboxy-terminal domain present in perilipin 1 involved in sequestering ABHD5 and suppressing basal lipolysis.

Proc Natl Acad Sci U S A 2014 Jun 9;111(25):9163-8. Epub 2014 Jun 9.

University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom

Lipid droplets (LDs) are a conserved feature of most organisms. Vertebrate adipocytes have evolved to efficiently store and release lipids for the whole organism from a single droplet. Perilipin 1, the most abundant lipid-coat protein in adipocytes, plays a key role in regulating lipolysis. In other tissues such as liver and muscle, LDs serve very different biological functions, buffering surplus lipids for subsequent oxidation or export. These tissues express perilipins 2 or 3, rather than perilipin 1. We sought to understand the role of perilipins 2 and 3 in regulating basal lipolysis. Bimolecular fluorescence complementation studies suggested that whereas perilipin 1 prevents the activation of adipose tissue triacylglycerol lipase by its coactivator, AB-hydrolase domain containing-5 (ABHD5), perilipins 2 and 3 do so less effectively. These differences are mediated by a conserved region within the carboxy terminus of perilipin 1 that binds and stabilizes ABHD5 by retarding its degradation by the proteosome. Chimeric proteins generated by fusing the carboxy terminus of perilipin 1 to the amino terminus of perilipins 2 or 3 stabilize ABHD5 and suppress basal lipolysis more effectively than WT perilipins 2 or 3. Furthermore, knockdown of perilipin 1 in adipocytes leads to replacement of perilipin 2 on LDs. In these cells we observed reduced ABHD5 expression and LD localization and a corresponding increase in basal lipolysis. Collectively these data suggest that whereas perilipin 1 potently suppresses basal lipolysis in adipocytes, perilipins 2 and 3 facilitate higher rates of basal lipolysis in other tissues where constitutive traffic of fatty acids via LDs is a necessary step in their metabolism.
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http://dx.doi.org/10.1073/pnas.1318791111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4078844PMC
June 2014

Mutations disrupting the Kennedy phosphatidylcholine pathway in humans with congenital lipodystrophy and fatty liver disease.

Proc Natl Acad Sci U S A 2014 Jun 2;111(24):8901-6. Epub 2014 Jun 2.

University of Cambridge, Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom;

Phosphatidylcholine (PC) is the major glycerophospholipid in eukaryotic cells and is an essential component in all cellular membranes. The biochemistry of de novo PC synthesis by the Kennedy pathway is well established, but less is known about the physiological functions of PC. We identified two unrelated patients with defects in the Kennedy pathway due to biallellic loss-of-function mutations in phosphate cytidylyltransferase 1 alpha (PCYT1A), the rate-limiting enzyme in this pathway. The mutations lead to a marked reduction in PCYT1A expression and PC synthesis. The phenotypic consequences include some features, such as severe fatty liver and low HDL cholesterol levels, that are predicted by the results of previously reported liver-specific deletion of murine Pcyt1a. Both patients also had lipodystrophy, severe insulin resistance, and diabetes, providing evidence for an additional and essential role for PCYT1A-generated PC in the normal function of white adipose tissue and insulin action.
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http://dx.doi.org/10.1073/pnas.1408523111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066527PMC
June 2014

GEI-8, a homologue of vertebrate nuclear receptor corepressor NCoR/SMRT, regulates gonad development and neuronal functions in Caenorhabditis elegans.

PLoS One 2013 6;8(3):e58462. Epub 2013 Mar 6.

Laboratory of Molecular Biology and Genetics, Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in Drosophila or C. elegans. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in C. elegans and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the gei-8 coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous gei-8 mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590189PMC
September 2013

Cystinosin, MPDU1, SWEETs and KDELR belong to a well-defined protein family with putative function of cargo receptors involved in vesicle trafficking.

Authors:
Vladimir Saudek

PLoS One 2012 17;7(2):e30876. Epub 2012 Feb 17.

University of Cambridge Metabolic Research Labs, Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, United Kingdom.

Classification of proteins into families based on remote homology often helps prediction of their biological function. Here we describe prediction of protein cargo receptors involved in vesicle formation and protein trafficking. Hidden Markov model profile-to-profile searches in protein databases using endoplasmic reticulum lumen protein retaining receptors (KDEL, Erd2) as query reveal a large and diverse family of proteins with seven transmembrane helices and common topology and, most likely, similar function. Their coding genes exist in all eukaryota and in several prokaryota. Some are responsible for metabolic diseases (cystinosis, congenital disorder of glycosylation), others are candidate genes for genetic disorders (cleft lip and palate, certain forms of cancer) or solute uptake and efflux (SWEETs) and many have not yet been assigned a function. Comparison with the properties of KDEL receptors suggests that the family members could be involved in protein trafficking and serve as cargo receptors. This prediction sheds new light on a range of biologically, medically and agronomically important proteins and could open the way to discovering the function of many genes not yet annotated. Experimental testing is suggested.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030876PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281891PMC
June 2012

The human lipodystrophy protein seipin is an ER membrane adaptor for the adipogenic PA phosphatase lipin 1.

Mol Metab 2012 26;2(1):38-46. Epub 2012 Dec 26.

University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK.

Disruption of the gene BSCL2 causes a severe, generalised lipodystrophy, demonstrating the critical role of its protein product, seipin, in human adipose tissue development. Seipin is essential for adipocyte differentiation, whilst the study of seipin in non-adipose cells has suggested a role in lipid droplet formation. However, its precise molecular function remains poorly understood. Here we demonstrate that seipin can inducibly bind lipin 1, a phosphatidic acid (PA) phosphatase important for lipid synthesis and adipogenesis. Knockdown of seipin during early adipogenesis decreases the association of lipin 1 with membranes and increases the accumulation of its substrate PA. Conversely, PA levels are reduced in differentiating cells by overexpression of wild-type seipin but not by expression of a mutated seipin that is unable to bind lipin 1. Together our data identify lipin as the first example of a seipin-interacting protein and reveals a novel molecular function for seipin in developing adipocytes.
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http://dx.doi.org/10.1016/j.molmet.2012.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757660PMC
September 2013

Human frame shift mutations affecting the carboxyl terminus of perilipin increase lipolysis by failing to sequester the adipose triglyceride lipase (ATGL) coactivator AB-hydrolase-containing 5 (ABHD5).

J Biol Chem 2011 Oct 12;286(40):34998-5006. Epub 2011 Jul 12.

Institute of Metabolic Science Metabolic Research Laboratories and the Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, United Kingdom.

Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740-748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.
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http://dx.doi.org/10.1074/jbc.M111.278853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186430PMC
October 2011

Endoplasmic reticulum stress-induced transcription factor, CHOP, is crucial for dendritic cell IL-23 expression.

Proc Natl Acad Sci U S A 2010 Oct 27;107(41):17698-703. Epub 2010 Sep 27.

Department of Medicine, University of Cambridge, School of Clinical Medicine, Addenbrookes Hospital, Cambridge CB2 0QQ, United Kingdom.

The endoplasmic reticulum (ER) stress response detects malfunctions in cellular physiology, and microbial pattern recognition receptors recognize external threats posed by infectious agents. This study has investigated whether proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress. Activation of ER stress, in combination with Toll-like receptor (TLR) agonists, markedly enhanced expression of mRNA of the unique p19 subunit of IL-23, and also significantly augmented secretion of IL-23 protein. These effects were not seen for IL-12 secretion. The IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation. Knockdown of CHOP in U937 cells significantly reduced the synergistic effects of TLR and ER stress on IL-23p19 expression, but did not affect expression of other LPS-responsive genes. The integration of ER stress signals and the requirement for CHOP in the induction of IL-23 responses was also investigated in a physiological setting: infection of myeloid cells with Chlamydia trachomatis resulted in the expression of CHOP mRNA and induced the binding of CHOP to the IL-23 promoter. Furthermore, knockdown of CHOP significantly reduced the expression of IL-23 in response to this intracellular bacterium. Therefore, the effects of pathogens and other environmental factors on ER stress can profoundly affect the nature of innate and adaptive immune responses.
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http://dx.doi.org/10.1073/pnas.1011736107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955096PMC
October 2010

Partial lipodystrophy and insulin resistant diabetes in a patient with a homozygous nonsense mutation in CIDEC.

EMBO Mol Med 2009 Aug;1(5):280-7

Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Madrid, Spain.

Lipodystrophic syndromes are characterized by adipose tissue deficiency. Although rare, they are of considerable interest as they, like obesity, typically lead to ectopic lipid accumulation, dyslipidaemia and insulin resistant diabetes. In this paper we describe a female patient with partial lipodystrophy (affecting limb, femorogluteal and subcutaneous abdominal fat), white adipocytes with multiloculated lipid droplets and insulin-resistant diabetes, who was found to be homozygous for a premature truncation mutation in the lipid droplet protein cell death-inducing Dffa-like effector C (CIDEC) (E186X). The truncation disrupts the highly conserved CIDE-C domain and the mutant protein is mistargeted and fails to increase the lipid droplet size in transfected cells. In mice, Cidec deficiency also reduces fat mass and induces the formation of white adipocytes with multilocular lipid droplets, but in contrast to our patient, Cidec null mice are protected against diet-induced obesity and insulin resistance. In addition to describing a novel autosomal recessive form of familial partial lipodystrophy, these observations also suggest that CIDEC is required for unilocular lipid droplet formation and optimal energy storage in human fat.
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http://dx.doi.org/10.1002/emmm.200900037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891108PMC
August 2009

Prevalence of loss-of-function FTO mutations in lean and obese individuals.

Diabetes 2010 Jan 15;59(1):311-8. Epub 2009 Oct 15.

CNRS 8090-Institute of Biology, Pasteur Institute, Lille, France.

Objective: Single nucleotide polymorphisms (SNPs) in intron 1 of fat mass- and obesity-associated gene (FTO) are strongly associated with human adiposity, whereas Fto(-/-) mice are lean and Fto(+/-) mice are resistant to diet-induced obesity. We aimed to determine whether FTO mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure-function relationships within FTO.

Research Design And Methods: We sequenced all coding exons of FTO in 1,433 severely obese and 1,433 lean individuals. We studied the enzymatic activity of selected nonsynonymous variants.

Results: We identified 33 heterozygous nonsynonymous variants in lean (2.3%) and 35 in obese (2.4%) individuals, with 8 mutations unique to the obese and 11 unique to the lean. Two novel mutations replace absolutely conserved residues: R322Q in the catalytic domain and R96H in the predicted substrate recognition lid. R322Q was unable to catalyze the conversion of 2-oxoglutarate to succinate in the presence or absence of 3-methylthymidine. R96H retained some basal activity, which was not enhanced by 3-methylthymidine. However, both were found in lean and obese individuals.

Conclusions: Heterozygous, loss-of-function mutations in FTO exist but are found in both lean and obese subjects. Although intron 1 SNPs are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that FTO has a role in energy balance, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese. Functional analyses of FTO mutations have given novel insights into structure-function relationships in this enzyme.
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http://dx.doi.org/10.2337/db09-0703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797938PMC
January 2010

Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations.

Am J Hum Genet 2009 Jul 25;85(1):106-11. Epub 2009 Jun 25.

INSERM U781 and Département de Génétique, Université Paris Descartes, Hôpital Necker-Enfants Malades, 75015 Paris, France.

FTO is a nuclear protein belonging to the AlkB-related non-haem iron- and 2-oxoglutarate-dependent dioxygenase family. Although polymorphisms within the first intron of the FTO gene have been associated with obesity, the physiological role of FTO remains unknown. Here we show that a R316Q mutation, inactivating FTO enzymatic activity, is responsible for an autosomal-recessive lethal syndrome. Cultured skin fibroblasts from affected subjects showed impaired proliferation and accelerated senescence. These findings indicate that FTO is essential for normal development of the central nervous and cardiovascular systems in human and establish that a mutation in a human member of the AlkB-related dioxygenase family results in a severe polymalformation syndrome.
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http://dx.doi.org/10.1016/j.ajhg.2009.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706958PMC
July 2009

Acute intermittent porphyria--impact of mutations found in the hydroxymethylbilane synthase gene on biochemical and enzymatic protein properties.

FEBS J 2009 Apr;276(7):2106-15

Department of Pediatrics, First School of Medicine, Charles University, Prague, Czech Republic.

Acute intermittent porphyria is an autosomal dominantly inherited disorder, classified as acute hepatic porphyria, caused by a deficiency of hydroxymethylbilane synthase (EC 2.5.1.61, EC 4.3.1.8, also known as porphobilinogen deaminase, uroporphyrinogen I synthase), the third enzyme in heme biosynthesis. Clinical features include autonomous, central, motor or sensory symptoms, but the most common clinical presentation is abdominal pain caused by neurovisceral crises. A diagnosis of acute intermittent porphyria is crucial to prevent life-threatening acute attacks. Detection of DNA variations by molecular techniques allows a diagnosis of acute intermittent porphyria in situations where the measurement of porphyrins and precursors in urine and faeces and erythrocyte hydroxymethylbilane synthase activity is inconclusive. In the present study, we identified gene defects in six Czech patients with acute intermittent porphyria, as diagnosed based on biochemical findings, and members of their families to confirm the diagnosis at the molecular level and/or to provide genetic counselling. Molecular analyses of the hydroxymethylbilane synthase gene revealed seven mutations. Four were previously reported: c.76C>T, c.77G>A, c.518G>A, c.771 + 1G>T (p.Arg26Cys, p.Arg26His, p.Arg173Gln). Three were novel mutations: c.610C>A, c.675delA, c.750A>T (p.Gln204Lys, p.Ala226ProfsX28, p.Glu250Asp). Of particular interest, one patient had two mutations (c.518G>A; c.610C>A), both located in exon 10 of the same allele. To establish the effects of the mutations on enzyme function, biochemical characterization of the expressed normal recombinant and mutated proteins was performed. Prokaryotic expression of the mutant alleles of the hydroxymethylbilane synthase gene revealed that, with the exception of the p.Gln204Lys mutation, all mutations resulted in little, if any, enzymatic activity. Moreover, the 3D structure of the Escherichia coli and human protein was used to interpret structure-function relationships for the mutations in the human isoform.
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http://dx.doi.org/10.1111/j.1742-4658.2009.06946.xDOI Listing
April 2009

Correlation between biochemical findings, structural and enzymatic abnormalities in mutated HMBS identified in six Israeli families with acute intermittent porphyria.

Blood Cells Mol Dis 2009 Mar-Apr;42(2):167-73. Epub 2009 Jan 12.

Department of Pediatrics and Center for Applied Genomics, 1st School of Medicine, Charles University, Prague, Czech Republic.

Mutations in the hydroxymethylbilane synthase (HMBS) gene are responsible for the inherited disorder of acute intermittent porphyria (AIP). AIP is diagnosed on the basis of characteristic clinical symptoms, elevated levels of urinary porphyrin precursors aminolevulinic acid (ALA) and porphobilinogen (PBG) and a decreased erythrocytic HMBS activity, although an identifiable HMBS mutation provides the ultimate proof for AIP. Six Israeli AIP families underwent biochemical and mutation analysis in order to establish an AIP diagnosis. Variability with respect to the ALA/PBG levels and HBMS activity was found among the index patients. Indeed, each family carried a unique mutation in the HMBS gene. A novel missense c.95G>C (p.R32P) was shown to be a de novo mutation in one family, along with five known mutations p.T59I, p.D178N, p.V215M, c.730_731delCT and c.982_983delCA identified in the rest of the families. Both R32P and D178N were expressed in a prokaryotic system. Recombinant p.R32P was enzymatically inactive as demonstrated by a <1% residual activity, whereas p.D178N possessed 81% of the activity of the wild type enzyme. However, the p.D178N mutant did display a shift in optimal pH and was thermo labile compared to the wild type. Among the four missense mutations, p.R32P and p.V215M had not only harmful effects on the enzyme in vitro but also were associated with high levels of ALA/PBG in patients. On the other hand, the in vitro effect of both p.T59I and p.D178N, and the impact of these mutations on the enzyme structure and function as interpreted by the 3-D structure of the Escherichia coli enzyme, were weaker than that of p.R32P and p.V215M. Concomitantly, patients carrying the p.T59I or p.D178N had normal or borderline increases in ALA/PBG concentrations although they presented characteristic clinical symptoms. These findings provided further insights into the causal relationship between HMBS mutations and AIP.
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http://dx.doi.org/10.1016/j.bcmd.2008.11.001DOI Listing
May 2009

The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.

Science 2007 Nov 8;318(5855):1469-72. Epub 2007 Nov 8.

Chemistry Research Laboratory and Oxford Centre for Integrative Systems Biology, University of Oxford, 12 Mansfield Road, Oxford, Oxon OX1 3TA, UK.

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.
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http://dx.doi.org/10.1126/science.1151710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668859PMC
November 2007

BIR-1, a Caenorhabditis elegans homologue of Survivin, regulates transcription and development.

Proc Natl Acad Sci U S A 2003 Apr 7;100(9):5240-5. Epub 2003 Apr 7.

Laboratory of Molecular Biology and Genetics, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, CZ-128 01 Prague 2, Czech Republic.

bir-1, a Caenorhabditis elegans inhibitor-of-apoptosis gene homologous to Survivin is organized in an operon with the transcription cofactor C. elegans SKIP (skp-1). Because genes arranged in operons are frequently linked functionally, we have asked whether BIR-1 also functions in transcription. bir-1 inhibition resulted in multiple developmental defects that overlapped with C. elegans SKIP loss-of-function phenotypes: retention of eggs, dumpy, movement defects, and lethality. bir-1 RNA-mediated interference decreased expression of several gfp transgenes and the endogenous genes dpy-7 and hlh-1. Immunoblot analysis revealed decreased phosphoacetylated histones in bir-1 RNA-mediated interference-treated worms. In a heterologous transfection system, BIR-1 augments thyroid hormone-regulated transcription and has an additive effect with SKIP. These results show that BIR-1 functions in the regulation of transcription and development.
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http://dx.doi.org/10.1073/pnas.0730770100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154329PMC
April 2003

Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase.

J Biol Chem 2003 Feb 6;278(8):6521-31. Epub 2002 Dec 6.

Department of Exploratory Research, Sanofi Synthelabo Recherche, 16 Rue d'Ankara, F-67080 Strasbourg, France.

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).
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http://dx.doi.org/10.1074/jbc.M209764200DOI Listing
February 2003