43 research outputs found
Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically
The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as “TRFS” probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes’ complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research
The effects of reminiscence on psychological well-being in older adults: a meta-analysis.
This paper presents the results of a meta-analysis to assess the effectiveness of reminiscence on psychological well-being across different target groups and treatment modalities. Fifteen controlled outcome studies were included. An overall effect size of 0.54 was found, indicating a moderate influence of reminiscence on life-satisfaction and emotional well-being in older adults. Life-review was found to have significantly greater effect on psychological well-being than simple reminiscence. In addition, reminiscence had significantly greater effect on community-dwelling adults than adults living in nursing homes or residential care. Other characteristics of participants or interventions were not found to moderate effects. It is concluded that reminiscence in general, but especially life review, are potentially effective methods for the enhancement of psychological well-being in older adults. However, a replication of effectiveness studies of the well-defined protocols is now warranted. © 2007 Taylor & Francis
Structural and functional characterization of Plasmodium falciparum nicotinic acid mononucleotide adenylyltransferase
Nicotinic acid mononucleotide adenylyltransferase (NaMNAT) is an indispensable enzyme for the synthesis of NAD and NAD phosphate. It catalyzes the adenylylation of nicotinic acid mononucleotide (NaMN) to yield nicotinic acid adenine dinucleotide (NaAD). Since NAD(H) and NAD phosphate(H) are essentially involved in metabolic and redox regulatory reactions, NaMNAT is an attractive drug target in the fight against bacterial and parasitic infections. Notably, NaMNAT of the malaria parasite Plasmodium falciparum possesses only 20% sequence identity with the homologous human enzyme. Here, we present for the first time the two X-ray structures of P. falciparum NaMNAT (PfNaMNAT)—in the product-bound state with NaAD and complexed with an α,β-non-hydrolizable ATP analog—the structures were determined to a resolution of 2.2 Å and 2.5 Å, respectively. The overall architecture of PfNaMNAT was found to be more similar to its bacterial homologs than its human counterparts although the PPHK motif conserved in bacteria is missing. Furthermore, PfNaMNAT possesses two cysteine residues within the active site that have not been described for any other NaMNATase so far and are likely to be involved in redox regulation of PfNaMNAT activity. Enzymatic studies and surface plasmon resonance data reveal that PfNaMNAT is capable of utilizing NaMN and nicotinamide mononucleotide with a slight preference for NaMN. Surprisingly, a comparison with the active site of Escherichia coli NaMNAT showed very similar architectures, despite different substrate preferences
Cyclic 5-Membered Disulfides Are Not Selective Substrates of Thioredoxin Reductase, but Are Opened Nonspecifically
The cyclic five-membered disulfide 1,2-dithiolane has been used as the key element in numerous chemical biology probes. Contradictory views of this disulfide populate the literature: some reports describe it as being nonspecifically reduced, others as a highly specific substrate for thioredoxin reductase (TrxR). We here show that 1,2-dithiolane probes are nonspecifically reduced by a broad
range of thiol reductants and redox-active proteins, and that
their cellular performance is barely affected by TrxR inhibition or knockout. We conclude that inhibitor screenings and "TRFS" probes that have used 1,2-dithiolanes as TrxRselective substrates should be treated with caution, and may need re-evaluation. Understanding 1,2-dithiolanes’ behaviour needs consideration of probe localisation and environmentdependent fluorescence, reduction-independent ring-opening polymerisation, thiol-dependent cellular uptake, and caution when applying thiophilic inhibitors. We present an approach controlling against assay misinterpretation with reducible probes, to ensure that future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research
Selective, Modular Probes for Thioredoxins Enabled by Rational Tuning of a Unique Disulfide Structure Motif
Specialised cellular networks of oxidoreductases coordinate the
dithiol/disulfide-exchange reactions that control metabolism, protein
regulation, and redox homeostasis. For probes to be selective for redox enzymes
and effector proteins (nM to µM concentrations), they must also be able to
resist nonspecific triggering by the ca. 50 mM background of non-catalytic
cellular monothiols. However, no such selective reduction-sensing systems have yet
been established. Here, we used rational structural design to independently vary
thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual
disulfide reduction trigger units designed for stability to monothiols. We integrated
the motifs into modular series of fluorogenic probes that release and activate
an arbitrary chemical cargo upon reduction, and compared their performance to that
of the literature-known disulfides. The probes were comprehensively screened
for biological stability and selectivity against a range of redox effector
proteins and enzymes. This design process delivered the first disulfide probes with
excellent stability to monothiols, yet high selectivity for the key
redox-active protein effector, thioredoxin. We anticipate that further
applications of these novel disulfide triggers will deliver unique probes targeting
cellular thioredoxins. We also anticipate that further tuning following this
design paradigm will deliver redox probes for other important dithiol-manifold redox
proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous
cellular redox systems.</p