Role of adipocyte lipid-binding protein (ALBP) and acyl-CoA binding protein (ACBP) in PPAR-mediated transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.     

The complete primary structure of late lactation protein from quokka (Setonix brachyurus)

The complete primary structure of the late lactation protein from the milk of quokka (Setonix brachyurus) is presented. The amino acid sequence was established by N-terminal sequence analysis of high-performance liquid chromatography purified intact protein and peptides isolated from chemical and enzymatic digests of the protein. The protein contains 158 residues including four cysteines. The sequence comparison with the tamar wallaby (Macropus eugenii) late lactation protein shows only five differences. The protein is identified as a new member of a novel late lactation protein family present in the milk of marsupials.     

Analysis of two linked genes coding for the acyl carrier protein (ACP) from Arabidopsis thaliana (columbia)

Two linked genes, A1 and A2, coding for nearly identical isoforms of the acyl carrier protein (ACP) were isolated from an Arabidopsis thaliana (columbia) genomic library and sequenced. The amino acids deduced from the nucleotide sequence of the two genes indicate they encode distinct transit peptides, but the mature proteins are the same except for residue 79. Both genes are predicted to contain three introns in similar positions, although they differ in sequence and length. The introns interrupt regions coding for a) the transit peptide, b) the junction of the transit peptide and mature protein, and c) the highly conserved domain surrounding serine 38 to which the phosphopantetheine is attached. Primer extension analysis indicates that at least A1 is active in young plants.     

Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris

Cost effective ¹³C/¹⁵N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The ¹³C/¹⁵N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut⁺) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (Mut^S) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for ¹³C-isotope labeling can be substantially reduced using the Mut⁺ strain compared to the Mut^S strain, as no ¹³C₃-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for ¹⁵N-labeling by an optimal supply of 10 g/L (¹⁵NH₄)₂SO₄ every 24 h.     

The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: Studies of its binding to F1 and its function

The binding of oligomycin sensitivity conferring protein (OSCP) to soluble beef-heart mitochondrial ATPase (F₁) has been investigated. OSCP forms a stable complex with F₁, and the F₁ · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted submitochondrial particles. The F₁ · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F₁ is inactivated by 90% or more. Both free F₁ and the F₁ · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their subunits; whether subunits are also lost is uncertain. The cold-treated F₁ · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted particles. OSCP also protects F₁ against modification of its subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F₁ cannot bind OSCP indicates that OSCP binds to the subunit of F₁ and that F₁ contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F₁ with the membrane sector of the mitochondrial ATPase system.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - OSCP oligomycin sensitivity conferring protein - SDS sodium dodecylsulfate This paper is dedicated to the memory of David E. Green—scholar, pioneer, visionary.     

The mitomycin C (MMC)-binding protein from MMC-producing microorganisms protects from the lethal effect of bleomycin: crystallographic analysis to elucidate the binding mode of the antibiotic to the protein

Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain.     

The serum- and glucocorticoid-induced protein kinase-1 (Sgk-1) mitochondria connection: Identification of the IF-1 inhibitor of the F1F0-ATPase as a mitochondria-specific binding target and the stress-induced mitochondrial localization of endogenous Sgk-1

The expression, localization and activity of the serum- and glucocorticoid-induced protein kinase, Sgk-1, are regulated by multiple hormonal and environmental cues including cellular stress. Biochemical fractionation and indirect immunofluorescence demonstrated that sorbitol induced hyperosmotic stress stimulated expression and triggered the localization of endogenous Sgk-1 into the mitochondria of NMuMG mammary epithelial cells. The immunofluorescence pattern of endogenous Sgk-1 was similar to that of a green fluorescent linked fusion protein linked to the N-terminal Sgk-1 fragment that encodes the mitochondrial targeting signal. In the presence or absence of cellular stress, exogenously expressed wild type Sgk-1 efficiently compartmentalized into the mitochondria demonstrating the mitochondrial import machinery per se is not stressed regulated. Co-immunoprecipitation and GST-pull down assays identified the IF-1 mitochondrial matrix inhibitor of the F₁F₀-ATPase as a new Sgk-1 binding partner, which represents the first observed mitochondrial target of Sgk-1. The Sgk-1/IF-1 interaction requires the 122-176 amino acid region within the catalytic domain of Sgk-1 and is pH dependent, occurring at neutral pH but not at slightly acidic pH, which suggests that this interaction is dependent on mitochondrial integrity. An in vitro protein kinase assay showed that the F₁F₀-ATPase can be directly phosphorylated by Sgk-1. Taken together, our results suggest that stress-induced Sgk-1 localizes to the mitochondria, which permits access to physiologically appropriate mitochondrial interacting proteins and substrates, such as IF-1 and the F₁F₀-ATPase, as part of the cellular stressed induced program.     

The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins (Reczek, P.R., Weissman, D., Huvos, P.E. and Fasman, G.D. (1982) Biochemistry 21, 993–1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in T_m, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.