Study of the Gc protein in several French population samples : genetic polymorphism by isoelectrofocusing and quantitative results]

The authors describe the results obtained in an examination by isoelectrofocusing of more than 1,000 serum samples from various regions in France. The autochthonous populations of the north side of the Pyrénées are characterized by a high frequency of the gene Gc2 and a very low frequency of the Gc1F gene. This distinguishes them from the other groups studied (southwestern, northwestern and eastern France). The quantitative analysis of the data emphasizes the difference. These results can only be explained by the interdependance of the genetic and metabolic factors.     

Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation

The optic atrophy 3 (OPA3) gene, which has no known homolog or biological function, is mutated in patients with hereditary optic neuropathies. Here, we identified OPA3 as an integral protein of the mitochondrial outer membrane (MOM), with a C-terminus exposed to the cytosol and an N-terminal mitochondrial targeting domain. By quantitative analysis, we demonstrated that overexpression of OPA3 significantly induced mitochondrial fragmentation, whereas OPA3 knockdown resulted in highly elongated mitochondria. Cells with mitochondria fragmented by OPA3 did not undergo spontaneous apoptotic cell death, but were significantly sensitized to staurosporine- and TRAIL-induced apoptosis. In contrast, overexpression of a familial OPA3 mutant (G93S) induced mitochondrial fragmentation and spontaneous apoptosis, suggesting that OPA3 mutations may cause optic atrophy via a gain-of-function mechanism. Together, these results indicate that OPA3, as an integral MOM protein, has a crucial role in mitochondrial fission, and provides a direct link between mitochondrial morphology and optic atrophy.     

Behaviour of intrinsically disordered proteins in protein–protein complexes with an emphasis on fuzziness

Intrinsically disordered proteins (IDPs) do not, by themselves, fold into a compact globular structure. They are extremely dynamic and flexible, and are typically involved in signalling and transduction of information through binding to other macromolecules. The reason for their existence may lie in their malleability, which enables them to bind several different partners with high specificity. In addition, their interactions with other macromolecules can be regulated by a variable amount of chemically diverse post-translational modifications. Four kinetically and energetically different types of complexes between an IDP and another macromolecule are reviewed: (1) simple two-state binding involving a single binding site, (2) avidity, (3) allovalency and (4) fuzzy binding; the last three involving more than one site. Finally, a qualitative definition of fuzzy binding is suggested, examples are provided, and its distinction to allovalency and avidity is highlighted and discussed.     

Characterization of the self-palmitoylation activity of the transport protein particle component Bet3

Bet3, a transport protein particle component involved in vesicular trafficking, contains a hydrophobic tunnel occupied by a fatty acid linked to cysteine 68. We reported that Bet3 has a unique self-palmitoylating activity. Here we show that mutation of arginine 67 reduced self-palmitoylation of Bet3, but the effect was compensated by increasing the pH. Thus, arginine helps to deprotonate cysteine such that it could function as a nucleophile in the acylation reaction which is supported by the structural analysis of non-acylated Bet3. Using fluorescence spectroscopy we show that long-chain acyl-CoAs bind with micromolar affinity to Bet3, whereas shorter-chain acyl-CoAs do not interact. Mutants with a deleted acylation site or a blocked tunnel bind to Pal-CoA, only the latter with slightly reduced affinity. Bet3 contains three binding sites for Pal-CoA, but their number was reduced to two in the mutant with an obstructed tunnel, indicating that Bet3 contains binding sites on its surface.     

Metallothionein: the multipurpose protein

Metallothioneins (MTs) are intracellular, low molecular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MTs have unique structural characteristics to give potent metal-binding and redox capabilities. A primary role has not been identified, and remains elusive, as further functions continue to be discovered. The most widely expressed isoforms in mammals, MT-1 and MT-2, are rapidly induced in the liver by a wide range of metals, drugs and inflammatory mediators. In teh gut and pancreas, MT responds mainly to Zn status. A brain isoform, MT-3, has a specific neuronal growth inhibitory activity, while MT-1 and MT-2 have more diverse functions related to their thiolate cluster structure. These include involvement in Zn homeostasis, protection against heavy metal (especially Cd) and oxidant damage, and metabolic regulation via Zn donation, sequestration and/or redox control. Use of mice with altered gene expression has enhance our understanding of the multifaceted role of MT, emphasised in this review.     

Bridging the molecular and biological functions of the oxysterol-binding protein family

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large eukaryotic gene family that transports and regulates the metabolism of sterols and phospholipids. The original classification of the family based on oxysterol-binding activity belies the complex dual lipid-binding specificity of the conserved OSBP homology domain (OHD). Additional protein- and membrane-interacting modules mediate the targeting of select OSBP/ORPs to membrane contact sites between organelles, thus positioning the OHD between opposing membranes for lipid transfer and metabolic regulation. This unique subcellular location, coupled with diverse ligand preferences and tissue distribution, has identified OSBP/ORPs as key arbiters of membrane composition and function. Here, we will review how molecular models of OSBP/ORP-mediated intracellular lipid transport and regulation at membrane contact sites relate to their emerging roles in cellular and organismal functions.     

Yolk protein differences between species ofDrosophila

Summary 1–3 major proteins considered as vitellins or yolk proteins are detected in mature eggs from differentDrosophila species by gel electrophoresis on gradient slab gels. Qualitative and quantiative differences are found even between closely related species. In heteroplastic transplantations, no correlation was found between the similarity or dissimilarity of the protein pattern on the one hand, and success of failure of egg development and vitellogenesis on the other hand.We thank Mr B. Barandun for excellent technical assistance. This investigation was supported by the Swiss National Science Foundation, grant No. 3.792.76.     

Structure-function relationships of the polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3′end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation. Received 16 August 2007; received after revision 18 September 2007; accepted 2 October 2007     

Oncogenic protein tyrosine kinases

FLT3, a member of the class III receptor tyrosine kinases (RTKs), is preferentially expressed on the cell surface of hematopoietic progenitors, and the ligand of FLT3 (FL) is expressed as a membrane-bound or soluble form by bone marrow stroma cells. It has been disclosed that FL-FLT3 interaction plays an important role in the maintenance, proliferation and differentiation of hematopoiesis. FLT3 is also expressed in a high proportion of acute myeloid leukemia (AML) and B-lineage acute lymphoblastic leukemia cells. Activating mutations of FLT3 are the most frequent genetic lesions in AML, and AML patients with FLT3 mutations have a worse prognosis than those with normal FLT3. Exploring the mechanism by which FLT3 mutations cause autoactivation and uncontrolled signaling might lead to a better understanding of how FLT3 becomes oncogenic and provide insights for the development of new drugs.     

Phylogenetic origin of LI-cadherin revealed by protein and gene structure analysis

The intestine specific LI-cadherin differs in its overall structure from classical and desmosomal cadherins by the presence of seven instead of five cadherin repeats and a short cytoplasmic domain. Despite the low sequence similarity, a comparative protein structure analysis revealed that LI-cadherin may have originated from a five-repeat predecessor cadherin by a duplication of the first two aminoterminal repeats. To test this hypothesis, we cloned the murine LI-cadherin gene and compared its structure to that of other cadherins. The intron-exon organization, including the intron positions and phases, is perfectly conserved between repeats 3–7 of LI-cadherin and 1–5 of classical cadherins. Moreover, the genomic structure of the repeats 1–2 and 3–4 is identical for LI-cadherin and highly similar to that of the repeats 1–2 of classical cadherins. These findings strengthen our assumption that LI-cadherin originated from an ancestral cadherin with five domains by a partial gene duplication event.Received 22 December 2003; received after revision 9 February 2004; accepted 27 February 2004     

Time-dependence of estradiol effects on protein synthesis in the rat neurohypophysis.

The incorporation of 3H-leucine into neurohypophyseal proteins was measured in vitro, 24 h after the administration of a single dose of estradiol (0.3 mug) to castrated female rats. Estradiol treatment caused a significant increase of 3H-leucine incorporation into proteins of the posterior lobe. The effects of estradiol depended largely upon time injection. Rats injected at 06.00 h, i.e., at the end of the dark period exhibited a 74% increase in protein synthesis, whereas rat injected at 14.00 h, i.e., at the middle of the light period only showed a 30% of increase.     

Medium- and short-chain dehydrogenase/reductase gene and protein families

Alcohol dehydrogenase 3 (ADH3) is highly conserved, ubiquitously expressed in mammals and involved in essential cellular pathways. A large active site pocket entails special substrate specificities: shortchain alcohols are poor substrates, while medium-chain alcohols and particularly the glutathione adducts S-hydroxymethylglutathione (HMGSH) and S-nitrosoglutathione (GSNO) are efficiently converted under concomitant use of NAD⁺/NADH. By oxidation of HMGSH, the spontaneous glutathione adduct of formaldehyde, ADH3 is implicated in the detoxification of formaldehyde. Through the GSNO reductase activity, ADH3 can affect the transnitrosation equilibrium between GSNO and S-nitrosated proteins, arguing for an important role in NO homeostasis. Recent findings suggest that ADH3-mediated GSNO reduction and subsequent product formation responds to redox states in terms of NADH availability and glutathione levels. Finally, a dual function of ADH3 is discussed in view of its potential implications for asthma.     

Demonstration of a cytosol protein binding cholesterol in the adrenal glands]

Characterization of a cytosolic protein of adrenal origin that binds cholesterol. Adrenal cytosol contains a thermostable protein which binds cholesterol specifically and non-covalently. The sedimentation coefficient of the complex is close to 3 S. The side chain of the cholesterol molecule is responsible for the binding specificity.     

Role of Sam68 as an adaptor protein in signal transduction

Sam68, the substrate of Src in mitosis, belongs to the family of RNA binding proteins. Sam68 contains consensus sequences to interact with other proteins via specific domains. Thus, Sam68 has various proline-rich sequences to interact with SH3 domain-containing proteins. Moreover, Sam68 also has a C-terminal domain rich in tyrosine residues that is a substrate for tyrosine kinases. Tyrosine phosphorylation of Sam68 promotes its interaction with SH2 containing proteins. The association of Sam68 with SH3 domain-containing proteins, and its tyrosine phosphorylation may negatively regulate its RNA binding activity. The presence of these consensus sequences to interact with different domains allows this protein to participate in signal transduction pathways triggered by tyrosine kinases. Thus, Sam68 participates in the signaling of T cell receptors, leptin and insulin receptors. In these systems Sam68 is tyrosine phosphorylated and recruited to specific signaling complexes. The participation of Sam68 in signaling suggests that it may function as an adaptor molecule, working as a dock to recruit other signaling molecules. Finally, the connection between this role of Sam68 in protein-protein interaction with RNA binding activity may connect signal transduction of tyrosine kinases with the regulation of RNA metabolism.Received 16 July 2004; received after revision 12 August 2004; accepted 18 August 2004     

Going vertical: functional role and working principles of the protein Inscuteable in asymmetric cell divisions

Coordinating mitotic spindle dynamics with cortical polarity is essential for stem cell asymmetric divisions. Over the years, the protein Inscuteable (Insc) has emerged as a key element determining the spindle orientation in asymmetric mitoses. Its overexpression increases differentiative divisions in systems as diverse as mouse keratinocytes and radial glial cells. To date, the molecular explanation to account for this phenotype envisioned Insc as an adaptor molecule bridging between the polarity proteins Par3:Par6:aPKC and the spindle pulling machines assembled on NuMA:LGN:Gαi. However, recent biochemical and structural data revealed that Insc and NuMA are competitive interactors of LGN, challenging the simplistic idea of a single apical macromolecular complex, and demanding a revision of the actual working principles of Insc.     

Species-dependent stereospecific serum protein binding of the oral anticoagulant drug phenprocoumon

Summary 13 mammalian species are classified into 3 clearcut groups with respect to the stereospecific serum proteinbinding of phenprocoumon: 2 groups showing opposed stereospecific binding characteristics and a 3rd group exhibiting no stereospecific binding. Structural differences in the albumin molecule account for these stereospecific differences in serum protein-binding.This research was supported by a grant from the Deutsche Forschungsgemeinschaft (Ja/185/6).     

Canonical protein inhibitors of serine proteases

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising -helical, sheet, and / proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.Received 28 March 2003; received after revision 12 May 2003; accepted 16 May 2003     

Species-dependent stereospecific serum protein binding of the oral anticoagulant drug phenprocoumon.

13 mammalian species are classified into 3 clearcut groups with respect to the stereospecific serum protein-binding of phenprocoumon: 2 groups showing opposed stereospecific binding characteristics and a 3rd group exhibiting no stereospecific binding. Structural differences in the albumin molecule account for these stereospecific differences in serum protein-binding.