Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence.

Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines.

Biogenesis of yeast Mia40 - uncoupling folding from import and atypical recognition features.

The discovery of the mitochondrial intermembrane space assembly (MIA) pathway was followed by studies that focused mainly on the typical small substrates of this disulfide relay system and the interactions between its two central partners: the oxidoreductase Mia40 and the FAD-protein Erv1. Recent studies have revealed that more complex proteins utilize this pathway, including Mia40 itself. In the present study, we dissect the Mia40 biogenesis in distinct stages, supporting a kinetically coordinated sequence of events, starting with (a) import and insertion through the Tim23 translocon, followed by (b) folding of the core of imported Mia40 assisted by the endogenous Mia40 and (c) final interaction with Erv1.

An intrinsically disordered domain has a dual function coupled to compartment-dependent redox control.

The functional role of unstructured protein domains is an emerging field in the frame of intrinsically disordered proteins. The involvement of intrinsically disordered domains (IDDs) in protein targeting and biogenesis processes in mitochondria is so far not known. Here, we have characterized the structural/dynamic and functional properties of an IDD of the sulfhydryl oxidase ALR (augmenter of liver regeneration) located in the intermembrane space of mitochondria.

Targeting and maturation of Erv1/ALR in the mitochondrial intermembrane space.

The interaction of Mia40 with Erv1/ALR is central to the oxidative protein folding in the intermembrane space of mitochondria (IMS) as Erv1/ALR oxidizes reduced Mia40 to restore its functional state. Here we address the role of Mia40 in the import and maturation of Erv1/ALR. The C-terminal FAD-binding domain of Erv1/ALR has an essential role in the import process by creating a transient intermolecular disulfide bond with Mia40.

A novel intermembrane space-targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding.

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity.

MIA40 is an oxidoreductase that catalyzes oxidative protein folding in mitochondria.

MIA40 has a key role in oxidative protein folding in the mitochondrial intermembrane space. We present the solution structure of human MIA40 and its mechanism as a catalyst of oxidative folding. MIA40 has a 66-residue folded domain made of an alpha-helical hairpin core stabilized by two structural disulfides and a rigid N-terminal lid, with a characteristic CPC motif that can donate its disulfide bond to substrates.

Plasticity and expanding complexity of the hepatic transcription factor network during liver development.

Cross-regulatory cascades between hepatic transcription factors have been implicated in the determination of the hepatic phenotype. Analysis of recruitments to regulatory regions and the temporal and spatial expression pattern of the main hepatic regulators during liver development revealed a gradual increase in complexity of autoregulatory and cross-regulatory circuits. Within these circuits we identified a core group of six transcription factors, which regulate the expression of each other and the expression of other downstream hepatic regulators.

Regulation of hepatic metabolic pathways by the orphan nuclear receptor SHP.

SHP (small heterodimer partner) is an important component of the feedback regulatory cascade, which controls the conversion of cholesterol to bile acids. In order to identify the bona fide molecular targets of SHP, we performed global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing SHP in the liver. We demonstrate that SHP affects genes involved in diverse biological pathways, and in particular, several key genes involved in consecutive steps of cholesterol degradation, bile acid conjugation, transport and lipogenic pathways.

Acetylation regulates transcription factor activity at multiple levels.

CREB-binding protein (CBP) possesses an intrinsic acetyltransferase activity capable of acetylating nucleosomal histones as well as several nonhistone proteins. Here, it is shown that CBP can acetylate hepatocyte nuclear factor-4 (HNF-4), a member of the nuclear hormone receptor family, at lysine residues within the nuclear localization sequence. CBP-mediated acetylation is crucial for the proper nuclear retention of HNF-4, which is otherwise transported out to the cytoplasm via the CRM1 pathway.

Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins.

Hepatocyte nuclear factor-1 (HNF-1) plays an important role in the regulation of a large number of genes expressed in the liver, kidney, and pancreatic beta-cells. In exploring the molecular mechanism involved in HNF-1-dependent gene activation in the in vivo chromatin context, we found that HNF-1 can physically interact with the histone acetyltransferases (HATs) CREB-binding protein (CBP), p300/CBP-associated factor (P/CAF), Src-1, and RAC3. The transcriptional activation potential of HNF-1 on a genome integrated promoter was strictly dependent on the synergistic action of CBP and P/CAF, which can independently interact with the N-terminal and C-terminal domain of HNF-1, respectively.

Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4.

Hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of a large number of liver-specific genes. We report here the isolation of three HNF-4 isoforms from a human liver cDNA library. hHNF-4A and hHNF-4B, differing by the insertion of 10 amino acids in the C-terminal region, have been previously identified in mouse, rat and human liver.

Transcriptional regulation of the apolipoprotein A-IV gene involves synergism between a proximal orphan receptor response element and a distant enhancer located in the upstream promoter region of the apolipoprotein C-III gene.

Apolipoprotein A-IV expression is limited to intestinal and hepatic cells, suggesting a tissue specific transcriptional regulation of its gene. To investigate the mechanism controlling apo A-IV transcription we have analysed its promoter region by in vitro DNA binding and transient transfection experiments. DNase I footprinting analysis of the proximal promoter with rat liver nuclear extracts revealed four protected regions: AIVA (-32 to -22), AIVB (-84 to -42), AIVC (-148 to -92) and AIVD (-274 to -250).