NAD binding by human CD38 analyzed by Trp189 fluorescence.

The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca mobilizing second messengers ADP-ribose (ADPR), 2'-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189.

2'-Deoxyadenosine 5'-diphosphoribose is an endogenous TRPM2 superagonist.

Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2.

Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349.

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca-permeable non-selective cation channel activated by the binding of adenosine 5'-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown.

Structure-activity relationship of adenosine 5'-diphosphoribose at the transient receptor potential melastatin 2 (TRPM2) channel: rational design of antagonists.

Adenosine 5'-diphosphoribose (ADPR) activates TRPM2, a Ca(2+), Na(+), and K(+) permeable cation channel. Activation is induced by ADPR binding to the cytosolic C-terminal NudT9-homology domain. To generate the first structure-activity relationship, systematically modified ADPR analogues were designed, synthesized, and evaluated as antagonists using patch-clamp experiments in HEK293 cells overexpressing human TRPM2.

Functional cooperation between CREM and GCNF directs gene expression in haploid male germ cells.

Cellular differentiation and development of germ cells critically depend on a coordinated activation and repression of specific genes. The underlying regulation mechanisms, however, still lack a lot of understanding. Here, we describe that both the testis-specific transcriptional activator CREMtau (cAMP response element modulator tau) and the repressor GCNF (germ cell nuclear factor) have an overlapping binding site which alone is sufficient to direct cell type-specific expression in vivo in a heterologous promoter context.

8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist.

cADPR (cyclic ADP-ribose) is a universal Ca(2+) mobilizing second messenger. In T-cells cADPR is involved in sustained Ca(2+) release and also in Ca(2+) entry. Potential mechanisms for the latter include either capacitative Ca(2+) entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2).

The role of thyroid hormone receptor DNA binding in negative thyroid hormone-mediated gene transcription.

Thyroid hormone 3,3',5-tri-iodothyronine (T3) regulates gene expression in a positive and negative manner. Here, we analyzed the regulation of a positively (mitochondrial glycerol-3-phosphate dehydrogenase) and negatively T3-regulated target gene (TSHalpha). Thyroid hormone receptor (TR) activates mGPDH but not TSH promoter fragments in a mammalian one-hybrid assay.

T3-mediated expression of PGC-1alpha via a far upstream located thyroid hormone response element.

Thyroid hormone (T3) has a profound influence on normal development, differentiation and metabolism. T3 induces complex gene expression patterns raises the question of how these expression patterns might be regulated. Since the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) induces very similar cellular energy metabolic pathways, we investigated the molecular mechanism of T3 regulation of PGC-1alpha.

T3-mediated gene expression is independent of PGC-1alpha.

Thyroid hormone (T3) has a profound influence on normal development, differentiation and metabolism, processes which are known to be regulated by the transcriptional coactivator PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator-1alpha). Since T3 rapidly induces PGC-1alpha expression, we investigated whether reduced PGC-1alpha levels lead to alterations in T3-mediated gene expression patterns. Using RNA interference, we reduced PGC-1alpha mRNA to approximately 10% of its initial concentration in rat pituitary GC cells.

Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b.

TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings.