Yeast two-hybrid screens imply that GGNBP1, GGNBP2 and OAZ3 are potential interaction partners of testicular germ cell-specific protein GGN1

Gametogenetin (Ggn) is a testicular germ cell-specific gene specifically expressed from late pachytene spermatocytes through round spermatids. The function of gametogenetin protein 1 (GGN1) remains unknown. Here, we used the yeast two-hybrid approach to look for more GGN1 interacting proteins. We found that gametogenetin binding protein 1 (GGNBP1), gametogenetin binding protein 2 (GGNBP2) and ornithine decarboxylase antizyme 3 (OAZ3) were potential GGN1 interaction partners. We determined the regions mediating the interactions and further showed the interactions between the proteins in mammalian cells by colocalization and coimmunoprecipitation experiments. Our work suggested that GGN1, GGNBP1, GGNBP2 and OAZ3 could be involved in a common process associated with spermatogenesis.     

线粒体Ca^2＋转运与细胞代谢调节

线粒体具有一套完整的Ｃａ＾２＋转运系统,包括两条摄取途径和三条释放途径。生理条件下,它们在细胞胞质与线粒体钙稳态维持以及细胞能量代谢中起重要作用,线粒体从胞质摄取的Ｃａ＾２＋可激活某些Ｃａ＾２＋敏感的呼吸酶和代谢过程。病理条件下,线粒体Ｃａ＾２＋转运发生紊乱,通过线粒体通透性转换导致细胞坏死或凋亡。     

Nitrogen oxide: regulation of the cell metabolism without involvement of the cellular receptor system

A review of a new important trend in the research of regulation of cell metabolism by nitrogen oxide is presented.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

Oxysterol-binding proteins: Sterol and phosphoinositide sensors coordinating transport,signaling and metabolism

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation.The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes.     

Role of acylCoA binding protein in acylCoA transport,metabolism and cell signaling

Long chain acylCoA esters (LCAs) act both as substrates and intermediates in intermediary metabolism and as regulators in various intracellular functions. AcylCoA binding protein (ACBP) binds LCAs with high affinity and is believed to play an important role in intracellular acylCoA transport and pool formation and therefore also for the function of LCAs as metabolites and regulators of cellular functions [1]. The major factors controlling the free concentration of cytosol long chain acylCoA ester (LCA) include ACBP [2], sterol carrier protein 2 (SCP2) [3] and fatty acid binding protein (FABP) [4]. Additional factors affecting the concentration of free LCA include feed back inhibition of the acylCoA synthetase [5], binding to acylCoA receptors (LCA-regulated molecules and enzymes), binding to membranes and the activity of acylCoA hydrolases [6].     

Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus

Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnB-GlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.     

The 14-3-3 proteins in regulation of cellular metabolism

Thirty years ago, it was discovered that 14-3-3 proteins could activate enzymes involved in amino acid metabolism. In the following decades, 14-3-3s have been shown to be involved in many different signaling pathways that modulate cellular and whole body energy and nutrient homeostasis. Large scale screening for cellular binding partners of 14-3-3 has identified numerous proteins that participate in regulation of metabolic pathways, although only a minority of these targets have yet been subject to detailed studies. Because of the wide distribution of potential 14-3-3 targets and the resurging interest in metabolic pathway control in diseases like cancer, diabetes, obesity and cardiovascular disease, we review the role of 14-3-3 proteins in the regulation of core and specialized cellular metabolic functions. We cite illustrative examples of 14-3-3 action through their direct modulation of individual enzymes and through regulation of master switches in cellular pathways, such as insulin signaling, mTOR- and AMP dependent kinase signaling pathways, as well as regulation of autophagy. We further illustrate the quantitative impact of 14-3-3 association on signal response at the target protein level and we discuss implications of recent findings showing 14-3-3 protein membrane binding of target proteins.     

Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.     

Glutamate transport and cellular glutamine metabolism: regulation in LLC-PK1 vs. LLC-PK1-F+ cell lines

The glutamate (Glu) transporter may modulate cellular glutamine(Gln) metabolism by regulating both the rates of hydrolysis andsubsequent conversion of Glu to -ketoglutarate andNH⁺₄. By delivering Glu, a competitiveinhibitor of Gln for the phosphate-dependent glutaminase (PDG) as wellas an acid-load activator of glutamate dehydrogenase (GDH) flux, thetransporter may effectively substitute extracellularly generated Glufrom the -glutamyltransferase for that derived intracellularly fromGln. We tested this hypothesis in two closely related porcine kidneycell lines, LLC-PK₁ and LLC-PK₁-F⁺,the latter selected to grow in the absence of glucose, relying on Glnas their sole energy source. Both cell lines exhibited PDG suppressionas the result of Glu uptake while disrupting the extracellularL-Glu uptake, withD-aspartate-acceleratedintracellular Glu formation coupled primarily to the ammoniagenicpathway (GDH). Conversely, enhancing the extracellular Glu formationwith p-aminohippurate and Glu uptakesuppressed intracellular Gln hydrolysis whileNH⁺₄ formation from Glu increased. Thus theseresults are consistent with the transporter's dual role in modulatingboth PDG and GDH flux. Interestingly, PDG flux was actually higher inthe Gln-adapted LLC-PK₁-F⁺cell line because of a two- to threefold enhancement in Gln uptake despite greater Glu uptake than in the parentalLLC-PK₁ cells, revealing theimportance of both Glu and Gln transport in the modulation of PDG flux.Nevertheless, when studied at physiological Gln concentration, PDG fluxfalls under tight Glu transporter control as Gln uptake decreases,suggesting that cellular Gln metabolism may indeed be under Glutransporter control in vivo.

     

Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.     

Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiquitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation-induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.     

Protective effects of riluzole on dopamine neurons: involvement of oxidative stress and cellular energy metabolism

Riluzole is neuroprotective in patients with amyotrophic lateral sclerosis and may also protect dopamine (DA) neurons in Parkinson's disease. We examined the neuroprotective potential of riluzole on DA neurons using primary rat mesencephalic cultures and human dopaminergic neuroblastoma SH-SY5Y cells. Riluzole (up to 10 microM:) alone affected neither the survival of DA neurons in primary cultures nor the growth of SH-SY5Y cells after up to 72 h. Riluzole (1-10 microM:) dose-dependently reduced DA cell loss caused by exposure to MPP(+) in both types of cultures. These protective effects were accompanied by a dose-dependent decrease of intracellular ATP depletion caused by MPP(+) (30-300 microM:) in SH-SY5Y cells without affecting intracellular net NADH content, suggesting a reduction of cellular ATP consumption rather than normalization of mitochondrial ATP production. Riluzole (1-10 microM:) also attenuated oxidative injury in both cell types induced by exposure to L-DOPA and 6-hydroxydopamine, respectively. Consistent with its antioxidative effects, riluzole reduced lipid peroxidation induced by Fe(3+) and L-DOPA in primary mesencephalic cultures. Riluzole (10 microM) did not alter high-affinity uptake of either DA or MPP(+). However, in the same cell systems, riluzole induced neuronal and glial cell death with concentrations higher than those needed for maximal protective effects (> or =100 microM:). These data demonstrate that riluzole has protective effects on DA neurons in vitro against neuronal injuries induced by (a) impairment of cellular energy metabolism and/or (b) oxidative stress. These results provide further impetus to explore the neuroprotective potential of riluzole in Parkinson's disease.     

Hormone regulation of cellular metabolism: Interplay of membrane transport and consecutive enzymic reaction

The regulatory effect of hormones on a steady-state process that consists of the mediated transport of a substrate across a membrane and a consecutive enzymic reaction is examined theoretically. The regulation of such a process depends not only on the effect of the hormone on the transport system, but also on the kinetic parameters of the reaction. The rate of metabolism can both increase and decrease due to a hormonal increase in the transport capacity, the affinity of the carrier to the substrate or the amount of energy involved, although the rate of mediated transport per se is always enhanced by such effects. Substrate inhibition of the enzyme can lead to multiple steady states and, thus, amplify hormone action. The quantitative results are believed to give insight into the hormonal regulation of both cellular uptake and intracellular metabolism.     

Carbonyl modified proteins in cellular regulation, aging, and disease

The oxidative modification of proteins by reactive species is implicated in the etiology or progression of a panoply of disorders and diseases. The level of these modified molecules can be quantitated by measurement of the protein carbonyl content, which has been shown to increase in a variety of diseases and processes, notably during aging. For the most part, oxidatively modified proteins are not repaired and must be removed by proteolytic degradation, a process which normally proceeds very efficiently, from microorganisms to mammals. In eukaryotes, removal is usually carried out by the proteosome, which selectively degrades oxidatively modified proteins, whether they be damaged by reactive oxygen species or specifically oxidized by cellular regulatory processes. The molecular deficiencies that cause accumulation of oxidatively modified proteins are not identified, but regardless of cause, the accumulation is likely to disrupt normal cellular function.