Phosphatidylinositol Transfer Protein Beta Research Articles

PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

Intracellular transport among organellar compartments occurs in two general ways, by membrane-bound carriers or membrane contacts. Specific circumstances that involve the coordination of these two modes of transport remain to be defined. Studying Coat Protein I (COPI) transport, we find that phosphatidylcholine with short acyl chains (sPC) is delivered through membrane contact from the endoplasmic reticulum (ER) to sites of COPI vesicle formation at the Golgi to support the fission stage. Phosphatidylinositol transfer protein beta (PITPβ) plays a key role in this process, with the elucidation of this role advancing a new understanding of how PITPβ acts, providing a mechanistic understanding of a specific circumstance when vesicular transport requires membrane contact, and contributing to a basic understanding of how transport carriers in a model intracellular pathway are formed.

Rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol counter-current for the formation of replication compartments at the ER-Golgi interface.

Similar to other positive-strand RNA viruses, rhinovirus, the causative agent of the common cold, replicates on a web of cytoplasmic membranes, orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of the replication membranes and complexes are poorly understood. We show that rhinovirus replication depends on host factors driving phosphatidylinositol 4-phosphate (PI4P)-cholesterol counter-currents at viral replication membranes. Depending on the virus type, replication required phosphatidylinositol 4-kinase class 3beta (PI4K3b), cholesteryl-esterase hormone-sensitive lipase (HSL) or oxysterol-binding protein (OSBP)-like 1, 2, 5, 9, or 11 associated with lipid droplets, endosomes, or Golgi. Replication invariably required OSBP1, which shuttles cholesterol and PI4P between ER and Golgi at membrane contact sites. Infection also required ER-associated PI4P phosphatase Sac1 and phosphatidylinositol (PI) transfer protein beta (PITPb) shunting PI between ER-Golgi. These data support a PI4P-cholesterol counter-flux model for rhinovirus replication.

Proteomic profile of pre - B2 lymphoblasts from children with acute lymphoblastic leukemia (ALL) in relation with the translocation (12; 21).

BackgroundUntil now, the major prognostic factors for pediatric acute lymphoblastic leukemia (ALL), age, white blood cell count and chromosomal alterations are initially taken into account for the risk stratification of patients. In the light of protein marker studies to classify subtypes of Acute Myeloblastic Leukemia efficiently, we have compared the lymphoblastes proteome in Childhood ALL in accordance with the presence of t(12;21), indicator of good prognosis, usually.MethodsProtein expression in pre-B2 lymphoblastic cells, collected from residual bone marrow cells after diagnostic procedures, was analyzed using two dimensional gel electrophoresis protocol. Protein spots whose average normalized volumes were statistically different in the two patients groups (n = 13; student t test p < 0.01), were excised. Tryptic peptides were then analyzed using a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a HPLC-chip cube interface. The tandem mass spectrometry peak lists extracted using the DataAnalysis program, were compared with the protein database Mascot Daemon.ResultsWe focused on twelve spots corresponding to sixteen identified candidate proteins among the 26 found differentially expressed (p ≤ 0.05) regarding the presence of t(12;21). Among over expressed proteins, two proteins were implicated in cellular growth arrest (i.e. calponine 2, p ≤ 0.001 and phosphatidylinositol transfer protein beta, p ≤ 0.001) in accordance with good prognosis, while two other proteins favored cell cycle proliferation (i.e. methionine adenosyl transferase 2β, p ≤ 0.005 and heterogeneous nuclear ribonucleo-proteins A2 p ≤ 0.01) and could therefore be good marker candidates of aggressiveness. Level of expression of proteasome subunit beta type-2 (p ≤ 0.01) and protein casein kinase 2α (p ≤ 0.01) which both favored apoptosis, deubiquitinating enzyme OTUB1 (p ≤ 0.05) and MLL septin-like fusion protein MSF-B, septin 9 i4 (p ≤ 0.01) were in accord with a good prognosis related to t(12;21) lymphoblasts.ConclusionBy drawing up the protein map of leukemic cells, these new data identified marker candidates of leukemic aggressiveness and new t(12;21) patients subgroups. These preliminary results will be in the near future confirmed by using a larger sample of pre-B2 childhood ALLs from national lymphoblastic cell collections.

Toxicoproteomic analysis of bovine aortic endothelial cell under exposure to cigarette smoking extracts

The major health implications of exposure to cigarette smoke that leads to disease development are still being explored. The overall protein expression pattern of bovine aortic endothelial cell (BAEC) has been studied during the treatment with cigarette smoke extract (CSE) by using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Out of an average 950 spots in silver-stained gels, the synthetic levels of 4 proteins were significantly increased, whereas 7 proteins were decreased. The identified proteins have been found to be involved in anti-inflammation (Apolipoprotein A1), energy metabolism (ATP synthase subunit d), signaling pathway (Acetyl-CoA carboxylase; GTP-binding protein), oxidative stress response (Phosphatidylinositol transfer protein beta isoform; phosphatidylinositol transfer protein alpha isoform; Vascular endothelial growth factor), carcinogenesis (Insulin-like growth factor-binding protein 2; Visinin-like protein-1), exocytosis machinery (Synaptosomal-associated protein 25) and immune response (C4b-binding protein). A significantly different protein expression pattern between the control and the CSE-exposed BAEC has been observed and demonstrated on the synthesis level analysis. The systematic approach to the analysis of proteomic responses used as well as the detailed analysis results has been useful in understanding the novel molecular mechanisms for cigarette smoke induced pathological changes. This will help in designing the systemic strategies for the development of new therapeutic approaches to smokerelated diseases.

In vivo dendritic cell depletion reduces breeding efficiency, affecting implantation and early placental development in mice

Implantation of mammalian embryos into their mother's uterus ensures optimal nourishment and protection throughout development. Complex molecular interactions characterize the implantation process, and an optimal synchronization of the components of this embryo-maternal dialogue is crucial for a successful reproductive outcome. In the present study, we investigated the role of dendritic cells (DC) during implantation process using a transgenic mouse system (DTRtg) that allows transient depletion of CD11c+ cells in vivo through administration of diphtheria toxin. We observed that DC depletion impairs the implantation process, resulting in a reduced breeding efficiency. Furthermore, the maturity of uterine natural killer cells at dendritic cell knockout (DCKO) implantation sites was affected as well; as demonstrated by decreased perforin expression and reduced numbers of periodic-acid-Schiff (PAS)-positive cells. This was accompanied by disarrangements in decidual vascular development. In the present study, we were also able to identify a novel DC-dependent protein, phosphatidylinositol transfer protein beta (PITPbeta), involved in implantation and trophoblast development using a proteomic approach. Indeed, DCKO mice exhibited substantial anomalies in placental development, including hypocellularity of the spongiotrophoblast and labyrinthine layers and reduced numbers of trophoblast giant cells. Giant cells also down-regulated their expression of two characteristic markers of trophoblast differentiation, placental lactogen 1 and proliferin. In view of these findings, dendritic cells emerge as possible modulators in the orchestration of events leading to the establishment and maintenance of pregnancy.

Acute and chronic effect of dietary phosphorus restriction on protein expression in young rat renal proximal tubules

Renal proximal tubules play a vital role in phosphorus (P) homeostasis. It is well known that dietary P restriction up-regulates the activities of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1-OHase), an enzyme that is involved in activation of vitamin D and thereby maintaining P balance. However, the mechanism involved in such regulation is not known. In the present study, we aim to identify proteins that might be involved in the renal adaptation to dietary P restriction using a proteomic approach. Renal proximal tubules were harvested from young rats fed either normal P diet or low P diet (LPD) for 1 to 7 days. Western blotting analysis of 1-OHase and signaling proteins in insulin-like growth factor I axis indicated an increase in expression of these proteins upon dietary P restriction. Using two-dimensional electrophoresis, we found that LPD reduced the total number of protein species expressed in renal proximal tubules. Differentially expressed proteins were analyzed and located using the software Melanie III, and their identities were found using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Our results showed that beta-actin, gamma-actin, major urinary protein, phosphatidylinositol transfer protein beta isoform, and G1/S-specific cyclin D3 are up-regulated and nonspecific lipid transfer protein is down-regulated by LPD.

The Golgi Localization of Phosphatidylinositol Transfer Protein β Requires the Protein Kinase C-dependent Phosphorylation of Serine 262 and Is Essential for Maintaining Plasma Membrane Sphingomyelin Levels

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein beta.

In order to study the in vivo function of the phosphatidylinositol transfer protein beta (PI-TPbeta), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPbeta. Two stable cell lines were isolated (SPIbeta2 and SPIbeta8) in which the levels of PI-TPbeta were increased 16- and 11-fold respectively. The doubling time of the SPIbeta cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPbeta expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-(3)H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [(3)H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [(3)H]SM in wt NIH3T3 cells was degraded, whereas the levels of [(3)H]SM in SPIbeta cells appeared to be unaffected. Since the release of [(3)H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIbeta cells, these results strongly suggest that breakdown of SM in SPIbeta cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [(3)H]SM in SPIbeta cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIbeta cells than in 50% [(3)H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIbeta cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIbeta cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPbeta plays an important role in restoring the steady-state levels of SM.