Biomedical subjects

Anne-Laure Perraud

Publications and source records attributed to Anne-Laure Perraud.

10 recordsLinked to original sources

PubMed2006

Metabolite of SIR2 reaction modulates TRPM2 ion channel.

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gating.

Adenosine Diphosphate Ribose↗

PubMed2005

The channel kinases TRPM6 and TRPM7 are functionally nonredundant.

TRPM7 and its closest homologue, TRPM6, are the only known fusions of an ion channel pore with a kinase domain. Deletion of TRPM7 in DT40 B-lymphocytes causes growth arrest, Mg(2+) deficiency, and cell death within 24-48 h. Amazingly, in analogy to TRPM6-deficient patients who can live a normal life if provided with a Mg(2+)-rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5-10 mm Mg(2+) concentrations in their extracellular medium. Here we have investigated the functional relationship between TRPM6 and TRPM7. We show that TRPM7 deficiency in DT40 cells cannot be complemented by heterologously expressed TRPM6. Nevertheless, both channels can influence each other's biological activity. Our data demonstrate that TRPM6 requires TRPM7 for surface expression in HEK-293 cells and also that TRPM6 is capable of cross-phosphorylating TRPM7 as assessed using a phosphothreonine-specific antibody but not vice versa. TRPM6 and TRPM7 coexpression studies in DT40 B-cells indicate that TRPM6 can modulate TRPM7 function. In conclusion, although TRPM6 and TRPM7 are closely related and deficiency in either one of these molecules severely affects Mg(2+) homeostasis regulation, TRPM6 and TRPM7 do not appear to be functionally redundant but rather two unique and essential components of vertebrate ion homeostasis regulation.

Cell Division↗

PubMed2004

Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels.

TRPM2 is a member of the transient receptor potential melastatin-related (TRPM) family of cation channels, which possesses both ion channel and ADP-ribose hydrolase functions. TRPM2 has been shown to gate in response to oxidative and nitrosative stresses, but the mechanism through which TRPM2 gating is induced by these types of stimuli is not clear. Here we show through structure-guided mutagenesis that TRPM2 gating by ADP-ribose and both oxidative and nitrosative stresses requires an intact ADP-ribose binding cleft in the C-terminal nudix domain. We also show that oxidative/nitrosative stress-induced gating can be inhibited by pharmacological reagents predicted to inhibit NAD hydrolysis to ADP-ribose and by suppression of ADP-ribose accumulation by cytosolic or mitochondrial overexpression of an enzyme that specifically hydrolyzes ADP-ribose. Overall, our data are most consistent with a model of oxidative and nitrosative stress-induced TRPM2 activation in which mitochondria are induced to produce free ADP-ribose and release it to the cytosol, where its subsequent accumulation induces TRPM2 gating via interaction within a binding cleft in the C-terminal NUDT9-H domain of TRPM2.

Adenosine Diphosphate Ribose↗

PubMed2004

Dual-function ion channel/protein kinases: novel components of vertebrate magnesium regulatory mechanisms.

Although magnesium is the dominant divalent intracellular cation and is required for the function of diverse types of enzymes that participate in virtually every cellular process, the molecular mechanisms that regulate its homeostasis are poorly understood. Electrophysiologic and biochemical investigations of a novel dual-function ion channel/kinase protein have recently converged with the identification of the gene locus for an autosomal recessive form of inherited hypomagnesemia to provide new insight into vertebrate magnesium regulatory mechanisms.

Animals↗

PubMed2003

Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7.

TRPM7 is a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 hr and eventually die. Here, we show that while TRPM7's kinase domain is not essential for activation of its channel, a functional coupling exists such that structural alterations of the kinase domain alter the sensitivity of channel activation to Mg(2+). Investigation of the relationship between Mg(2+) and the cell biological role of TRPM7 revealed that TRPM7-deficient cells become Mg(2+) deficient, that both the viability and proliferation of TRPM7-deficient cells are rescued by supplementation of extracellular Mg(2+), and that the capacity of heterologously expressed TRPM7 mutants to complement TRPM7 deficiency correlates with their sensitivity to Mg(2+). Overall, our results indicate that TRPM7 has a central role in Mg(2+) homeostasis as a Mg(2+) uptake pathway regulated through a functional coupling between its channel and kinase domains.

Adenosine Triphosphate↗

PubMed2003

Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation.

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.

Calcium↗

PubMed2002

NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase.

We have recently characterized the protein product of the human NUDT9 gene as a highly specific ADP-ribose (ADPR) pyrophosphatase. We now report an analysis of the human NUDT9 gene and its potential alternative transcripts along with detailed studies of the enzymatic properties and cell biological behavior of human NUDT9 protein. Our analysis of the human NUDT9 gene and twenty-two distinct cloned NUDT9 transcripts indicates that the full-length NUDT9 alpha transcript is the dominant form, and suggests that an alternative NUDT9 beta transcript occurs as the result of a potentially aberrant splice from a cryptic donor site within the first exon to the splice acceptor site of exon 2. Computer analysis of the predicted protein of the NUDT9 alpha transcript identified an N-terminal signal peptide or subcellular targeting sequence. Using green fluorescence protein tagging, we demonstrate that the predicted human NUDT9 alpha protein is targeted highly specifically to mitochondria, whereas the predicted protein of the NUDT9 beta transcript, which is missing this sequence, exhibits no clear subcellular localization. Investigation of the physical and enzymatic properties of NUDT9 indicates that it is functional as a monomer, optimally active at near neutral pH, and that it requires divalent metal ions and an intact Nudix motif for enzymatic activity. Furthermore, partial proteolysis of NUDT9 suggests that NUDT9 enzymes consist of two distinct domains: a proteolytically resistant C-terminal domain retaining essentially full specific ADPR pyrophosphatase activity and a proteolytically labile N-terminal portion that functions to enhance the affinity of the C-terminal domain for ADPR.

Amino Acid Sequence↗

PubMed2002

Analysis of O-acetyl-ADP-ribose as a target for Nudix ADP-ribose hydrolases.

The Sir2 family of NAD(+)-dependent histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life span extension, and chromosomal stability. Recent evidence has indicated that these proteins produce a novel metabolite O-acetyl-ADP-ribose (OAADPr) during deacetylation. Cellular studies have demonstrated that this metabolite exhibits biological effects when microinjected in living cells. However, the molecular targets of OAADPr remain to be identified. Here we have analyzed the ADP-ribose-specific Nudix family of hydrolases as potential in vivo metabolizing enzymes of OAADPr. In vitro, we found that the ADP-ribose hydrolases (yeast YSA1, mouse NudT5, and human NUDT9) cleaved OAADPr to the products AMP and acetylated ribose 5'-phosphate. Steady-state kinetic analyses revealed that YSA1 and NudT5 hydrolyzed OAADPr with similar kinetic constants to those obtained with ADP-ribose as substrate. In dramatic contrast, human NUDT9 was 500-fold less efficient (k(cat)/K(m) values) at hydrolyzing OAADPr compared with ADP-ribose. The inability of OAADPr to inhibit the reaction of NUDT9 with ADP-ribose suggests that NUDT9 binds OAADPr with low affinity, likely due to steric considerations of the additional acetylated-ribose moiety. We next explored whether Nudix hydrolytic activities against OAADPr could be observed in cell extracts from yeast and human. Using a detailed analysis of the products generated during the consumption of OAADPr in extracts, we identified two robust enzymatic activities that were not consistent with the known Nudix hydrolases. Instead, we identified cytoplasmic esterase activities that hydrolyze OAADPr to acetate and ADP-ribose, whereas a distinct activity residing in the nucleus is consistent with an OAADPr-specific acetyltransferase. These findings establish for the first time that select members of the ADP-ribose hydrolases are potential targets of OAADPr metabolism. However, the predominate endogenous activities observed from diverse cell extracts represent novel enzymes.

Animals↗

PubMed2002

Regulatory factors of Bordetella pertussis affecting virulence gene expression.

Most pathogenic bacteria encounter changing growth conditions during their infectious cycle and, accordingly, have to modulate gene expression to enable the efficient colonization of different environments outside or within their host organisms. In Bordetella pertussis the transcription of most virulence factors including several toxins and adhesins is regulated coordinately by the BvgAS two-component system. The molecular characterization of the BvgAS system revealed that it belongs to the small group of unorthodox two-component systems applying an obligate multistep phosphorelay. Moreover, despite the coordinated control of the virulence regulon, subtle differences in the regulation of individual virulence genes were observed which led to the identification of sophisticated mechanisms possibly engaged in fine tuning of virulence gene expression.

Bacterial Proteins↗

PubMedDate not supplied

TRPM2 Ca2+ permeable cation channels: from gene to biological function.

TRPM2 is a recently identified TRPM family cation channel which is unique among known ion channels in that it contains a C-terminal domain which is homologous to the NUDT9 ADP-ribose hydrolase and possesses intrinsic ADP-ribose hydrolase activity. Here, available information on the TRPM2 gene, transcripts, predicted protein products, and assembled multimeric channels is comprehensively reviewed and synthesized to highlight important areas for future work and provide insight into potential biological function(s) of TRPM2 channels.

Adenosine Diphosphate Ribose↗