Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.     

Preharvest application of exogenous abscisic acid (ABA) or an ABA analogue does not affect endogenous ABA concentration of onion bulbs

Bulb abscisic acid (ABA) concentration has been shown to decrease in stored onions, and onset of sprouting to occur at minimal ABA concentration. It was postulated that increasing prestorage bulb ABA concentration could increase storage life. Analogues of ABA that enhance biological activity and resist degradation are available and are becoming commercially viable. Exogenous ABA and an ABA analogue (8′-methylene ABA methyl ester; PBI-365) were applied separately as preharvest foliar sprays to six onion cultivars with varying storage potential. Quality indicators including pyruvate, total soluble solids and firmness were determined at regular intervals during storage. Neither ABA treatment increased endogenous bulb ABA concentration. Bulb ABA concentration decreased during storage and the onset of sprouting occurred at a minimal ABA concentration (ca. 50–120 ng g⁻¹ DW). This was followed by an increase in ABA concentration as sprout growth continued. No straightforward relationship between ABA and carbohydrate metabolism could be determined.     

Knocking down the diencephalic thyrotropin-releasing hormone precursor gene normalizes obesity-induced hypertension in the rat

We recently showed that diencephalic TRH may mediate the central leptin-induced pressor effect. Here, to study the role of TRH in obesity-induced hypertension (OIH), we used a model of OIH produced by a high-fat diet (HFD, 45 days) in male Wistar rats. After 4 wk, body weight and systolic arterial blood pressure (SABP) increased in HFD animals. Plasma leptin was correlated with peritoneal adipose tissue. Then, we treated OIH animals with an antisense oligodeoxynucleotide and small interfering (si)RNA against the prepro-TRH. Antisense significantly decreased diencephalic TRH content and SABP at 24 and 48 h posttreatment. Similar effects were observed with siRNA against prepro-TRH but for up to 4 wk. Conversely, vehicle, an inverted antisense sequence and siRNA against green fluorescence protein, produced no changes. SABP decrease seems to be owing to an inhibition of the obesity-enhanced sympathetic outflow but not to an alteration in thyroid status. Using a simple OIH model we demonstrated, for the first time, that central TRH participates in the hypertension induced by body weight gain probably through its well-known action on sympathetic activity. Thus the TRH-leptin interaction may contribute to the strong association between hypertension and obesity.     

Ammonium transport and CitAMT1 expression are regulated by light and sucrose in Citrus plants

Here the isolation and characterization of CitAMT1 cDNA from citrange Troyer (Citrus sinensis L. OsbeckxPoncirus trifoliata Blanco) is reported, suggesting that this belongs to the AMT gene family, which is involved in the high-affinity transport system (HATS). Results show that in Citrus plants, the HATS is much more dependent on the light conditions and C status of the roots than the low-affinity transport system. Most importantly, a strong correlation was found between the regulation of both HATS activity and CitAMT1 expression. CitAMT1 expression is sucrose-stimulated and may account for the regulation of NH(4)(+) HATS. Furthermore, a similar link was also recorded with photosynthetic activity in the shoots, suggesting that the variations in production and transport of photosynthates to the roots are responsible for the diurnal changes of both CitAMT1 expression and NH(4)(+) HATS activity. On the other hand, results indicate that the effect of stimulating light on CitAMT1 expression and NH(4)(+) HATS activity is independent of the circadian rhythm. Finally, CitAMT1 expression seems to be specifically stimulated by sucrose, suggesting that sucrose is a pivotal signal governing both assimilate partitioning from source organs and assimilate utilization in sink organs.     

Packing of transmembrane domain 2 of carnitine palmitoyltransferase-1A affects oligomerization and malonyl-CoA sensitivity of the mitochondrial outer membrane protein

The purpose of this study was to investigate the sequence-dependence of oligomerization of transmembrane domain 2 (TM2) of rat carnitine palmitoyltransferase 1A (rCPT1A), to elucidate the role of this domain in the function of the full-length enzyme. Oligomerization of TM2 was studied qualitatively using complementary genetic assays that facilitate measurement of helix-helix interactions in the Escherichia coli inner membrane, and multiple quantitative biophysical methods. The effects of TM2-mutations on oligomerization and malonyl-CoA inhibition of the full-length enzyme (expressed in the yeast Pichia pastoris) were quantified. Changes designed to disrupt close-packing of the GXXXG(A) motifs reduced the oligomeric state of the corresponding TM2 peptides from hexamer to trimer (or lower), a reduction also observed on mutation of the TM2 sequence in the full-length enzyme. Disruption of these GXXXG(A) motifs had a parallel effect on the malonyl-CoA sensitivity of rCPT1A, reducing the IC(50) from 30.3 ± 5.0 to 3.0 ± 0.6 μM. For all measurements, wild-type rCPT1A was used as a control alongside various appropriate (e.g., molecular mass) standards. Our results suggest that sequence-determined, TM2-mediated oligomerization is likely to be involved in the modulation of malonyl-CoA inhibition of CPT1A in response to short- and long-term changes in protein-protein and protein-lipid interactions that occur in vivo.     

Suppressor of cytokine signalling protein SOCS3 expression is increased at sites of acute and chronic inflammation

Treatment of cells with cytokines and growth factors leads to the synthesis of Suppressor of Cytokine Signalling (SOCS) proteins that act as potent negative regulators of signalling via the Jak/STAT pathway. We used immunohistochemistry to identify cells and pathologies where SOCS3 expression might influence acute and chronic inflammatory responses in human tissues. Epitope and GFP tagged SOCS3 fusion proteins were localised predominantly in the nucleus of transfected cells and a validated anti SOCS3 antiserum revealed the expression of SOCS3 in the nucleus and cytoplasm of macrophages, endothelial and epithelial cells in a wide range of normal tissues in tissue microarrays (n = 31 different tissues). Nuclear SOCS3 was only seen in cells expressing a high level of the protein. Comparative immunostaining of acute, chronically and granulomatously inflamed human tissues revealed higher levels of nuclear and cytoplasmic SOCS3 expression in inflamed than in corresponding normal tissues, particularly in recruited leukocyte populations, but also in epithelia. The staining appeared more intense, suggesting higher expression levels, in areas where inflammation was more acute, consistent with the time course of SOCS3 induction described in vitro. Expression of SOCS3 protein by leucocytes and other cell types in tissue sections could be a useful marker of cells undergoing acute or chronic stimulation by cytokines in vivo.