Cbl Enforces an SLP76-dependent Signaling Pathway for T Cell Differentiation

A signaling pathway involving ZAP-70, LAT, and SLP76 has been regarded as essential for receptor-driven T cell development and activation. Consistent with this model, mice deficient in SLP76 have a complete block at the double negative 3 stage of T cell development. Recently, however, it has been reported that inactivation of Cbl, a ubiquitin-protein isopeptide ligase, partially rescues T cell development in SLP76-deficient mice. To probe the influence of Cbl on domain-specific SLP76 functions, we reconstituted SLP76^-/- Cbl^-/- mice with Slp76 transgenes bearing mutations in each of three functional domains of SLP76 as follows: Y3F, in which the amino-terminal tyrosine residues of SLP76 were mutated, eliminating sites of SLP76 interaction with Vav, Nck, and Itk; Δ20, in which 20 amino acids in the proline-rich region of SLP76 were deleted, removing a binding site for Gads; and RK, in which arginine 448 of SLP76 was replaced by lysine, abolishing function of the Src homology 2 domain. Although each of these transgenes has been shown to partially rescue T cell development in SLP76^-/- mice, we report here that Cbl inactivation completely reverses the severe double negative 3 developmental block that occurs in SLP76-deficient mice expressing the Y3F transgene (Y3F mice) and partially rescues the defect in positive selection in T cell receptor transgenic Y3F mice, but in contrast fails to rescue thymic development of SLP76-deficient mice expressing the Δ20 or RK transgene. Rescue in SLP76^-/-Cbl^-/-Y3F double-positive thymocytes is associated with enhanced tyrosine phosphorylation of signaling molecules, including Lck, Vav, PLC-γ1, and ERKs, but not Itk, in response to T cell receptor stimulation. Thus, our data demonstrate that Cbl suppresses activation of a bypass signaling pathway and thereby enforces SLP76 dependence of early T cell development.T cell development proceeds through multiple stages that regulate the generation and selection of T cells whose T cell receptors (TCR)² have an appropriate range of affinity for peptides presented by major histocompatibility complex (MHC) molecules (1). Precursors give rise to immature CD4^-CD8^- double negative (DN) cells that can be further divided into DN1, DN2, DN3, and DN4 stages, distinguished by cell surface phenotype as well as by critical events, including expansion of DN3 cells that have successfully rearranged TCRβ and have expressed and signaled through the pre-TCR complex (2). DN3 cells differentiate to the DN4 and then CD4⁺CD8⁺ double-positive (DP) stage following pre-TCR signaling. DP thymocytes rearrange TCRα, express a mature TCRαβ receptor, and develop into mature CD4⁺CD8^- or CD4^-CD8⁺ single-positive (SP) cells through a process of positive and negative selection that is based on signaling through this mature TCR and selection of a T cell repertoire that is tolerant to self but capable of responding to foreign-peptide-MHC (pMHC) complexes (1, 3, 4). Finally, SP cells exit from the thymus as mature T cells capable of recognizing and responding to foreign antigens.The signals from pre-TCR and TCR, which determine the fate of developing thymocytes, have been intensely studied. Ligation of the TCR by pMHC complexes results in activation of a signaling cascade initiated by phosphorylation and activation of TCR-ζ, Lck, and ZAP-70, which in turn phosphorylate downstream targets, including LAT and SLP76. ZAP-70, LAT and SLP76 proteins (3) have been shown to be essential for thymocyte development by studies, including genetic manipulation in mice (5–8). There are essentially no detectable DP or SP thymocytes or peripheral T cells in LAT^-/- or SLP76^-/- mice, in which thymocyte development is blocked at the DN3 stage (5, 7). ZAP70^-/- thymocytes are blocked at the DP stage of T cell development, and ZAP70^-/- mice have very few SP thymocytes or peripheral T cells (6). These studies suggest that signal transduction required for early T cell development proceeds through a pathway that involves critical roles of multiple molecules, including ZAP-70, LAT, and SLP76.SLP76 consists of three functional domains as follows: an amino-terminal domain containing targets for tyrosine phosphorylation, a proline-rich region, and a carboxyl-terminal SH2 domain (9). The amino-terminal tyrosine residues (Tyr-112, Tyr-128, and Tyr-145) are phosphorylated by tyrosine kinases following TCR engagement, enabling SLP76 to interact with Vav, a Rho guanine nucleotide exchange factor, Nck, an adaptor protein, and Itk, a member of Tec family PTK. The proline-rich region of SLP76 has the capacity to bind Gads, a Grb2 homolog, which results in the recruitment of SLP76 to cell surface membrane lipid rafts through binding to LAT following TCR engagement. The carboxyl-terminal SH2 domain of SLP76 interacts with ADAP (adhesion and degranulation-promoting protein) (10) an adaptor protein, and HPK-1, a serine kinase (9). Reconstitution of SLP76-deficient mice with transgenes containing mutations in each of these domains has demonstrated that each region is required for normal thymocyte development (5, 8). Two groups have reconstituted SLP76-deficient mice with T cell-specific expression of wild-type and mutant SLP76 transgenes, including a mutant in which three tyrosine residues (Tyr-112, Tyr-128, and Tyr-145) in the amino-terminal domain of SLP76 were substituted by phenylalanines (Y3F); a mutant in which 20 amino acids (amino acids 224–244) in the proline-rich region of SLP76 were deleted (Δ20); and a mutant in which arginine 448 of SLP76 was replaced by lysine (RK) (11, 12). The profound defects in T cell development and activation that are observed in SLP76 knock-out mice are completely reversed by reconstitution with a wild-type SLP76 transgene. In contrast, however, reconstitution with SLP76 that has been mutated in any of its three functional domains only partially rescues T cell development in SLP76 knock-out mice.c-Cbl (Cbl) is a ubiquitin ligase and adaptor protein (regulator) with multiple domains that associate with multiple molecules involved in signal transduction (13). Thymocytes from Cbl knock-out mice have enhanced cell surface expression of TCR and CD3 in comparison with control mice (14, 15). In addition, it has been observed that phosphorylation of ZAP-70, LAT, and SLP76 is increased in Cbl^-/- mouse thymocytes (14, 15). Recently, we reported that inactivation of Cbl partially rescues T cell development in LAT and SLP76-deficient mice (16), and Myers et al. (17) reported that inactivation of Cbl partially rescues T cell development in ZAP-70-deficient mice. These observations indicate that Cbl mediates requirements for LAT, SLP76, and ZAP-70 by preventing signaling that is capable of supporting T cell differentiation independent of LAT, SLP76, or ZAP-70. However, the rescue of T cell development in these model systems is strikingly incomplete, failing to substantially reconstitute development through the pre-TCR-dependent DN3-DN4 transition and thus failing to generate normal numbers of DP or functionally mature SP thymocytes. These findings suggest that Cbl inactivation functions to enable pathways that are capable of bypassing some but not all of the requirements for ZAP-70, LAT, and SLP76 during T cell development. To define these signaling pathways, normally suppressed by Cbl, that can support T cell development, we assessed the ability of Cbl inactivation to rescue T cell development in the presence of Y3F, Δ20, or RK SLP76 mutant transgenes. In this study, we report that Cbl inactivation completely reverses the DN3-DN4 developmental defect and partially reverses alterations in positive selection in thymocytes of SLP76 knock-out mice reconstituted with the SLP76 mutant Y3F, which lacks amino-terminal phosphotyrosine residues. In contrast, Cbl inactivation has no effect on the thymic developmental defects observed in SLP76 knock-out mice reconstituted with Slp76 transgenes mutated in the proline-rich Gads-binding region (Δ20) or the carboxyl-terminal SH2 domain (RK). Biochemical studies revealed that rescue of development in SLP76^-/-Y3F thymocytes by inactivation of Cbl was marked by reversal of defects in tyrosine phosphorylation of multiple molecules, including Lck, Vav, PLC-γ1, and ERKs in response to TCR stimulation of DP thymocytes. Thus, Cbl normally enforces SLP76 dependence of T cell development by inhibiting an alternative pathway that may be independent of SLP76 association with Vav, Nck, and Itk (18).     

The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity

TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.     

Regulation of cyclin A localization downstream of Par-1 function is critical for the centrosome orientation checkpoint in Drosophila male germline stem cells

Male germline stem cells (GSCs) in Drosophila melanogaster divide asymmetrically by orienting the mitotic spindle with respect to the niche, a microenvironment that specifies stem cell identity. The spindle orientation is prepared during interphase through stereotypical positioning of the centrosomes. We recently demonstrated that GSCs possess a checkpoint ("the centrosome orientation checkpoint") that monitors correct centrosome orientation prior to mitosis to ensure an oriented spindle and thus asymmetric outcome of the division. Here, we show that Par-1, a serine/threonine kinase that regulates polarity in many systems, is involved in this checkpoint. Par-1 shows a cell cycle-dependent localization to the spectrosome, a germline-specific, endoplasmic reticulum-like organelle. Furthermore, the localization of cyclin A, which is normally localized to the spectrosome, is perturbed in par-1 mutant GSCs. Interestingly, overexpression of mutant cyclin A that does not localize to the spectrosome and mutation in hts, a core component of the spectrosome, both lead to defects in the centrosome orientation checkpoint. We propose that the regulation of cyclin A localization via Par-1 function plays a critical role in the centrosome orientation checkpoint.     

A Triglyceride‐Raising APOA5 Genetic Variant Is Negatively Associated With Obesity and BMI in the Chinese Population

Apolipoprotein A‐V (apo A‐V) exerts a potent triglyceride (TG)‐lowering effect through enhanced intravascular TG‐hydrolysis with increased uptake of TG‐derived free fatty acids into muscle and adipose tissue. Genetic variants in the APOA5 gene were strongly associated with plasma TG concentrations. The aim of this study was to examine whether APOA5 genetic variation was associated with obesity. We genotyped the missense c.553 G>T polymorphism (p.G185C) in the APOA5 gene in 1,085 Chinese (333 obese subjects and 752 nonobese controls). We analyzed the association between the c.553 G>T polymorphism and obesity and related metabolic phenotypes. The T allele at the c.553 G>T polymorphism was associated with higher plasma TG concentrations. Each additional T allele was associated with an increased TG concentration of 53.5 mg/dl (95% confidence interval (CI) 29.6–76.0, P < 0.0001). However, the T allele was associated lower risk of obesity (odds ratio (OR), 0.48; 95% CI 0.32–0.73, P = 0.0004). Each additional copy of the T allele was associated with a BMI decrease of 0.73 kg/m² (95% CI 0.26–1.16, P = 0.002), equivalent to 2.11 kg in a person 1.7 m tall. We may then conclude that the TG‐raising APOA5 genetic variant was associated with a decrease in BMI and reduced risk of obesity in the Chinese population.     

Decreased level of Nurr1 in heterozygous young adult mice leads to exacerbated acute and long-term toxicity after repeated methamphetamine exposure

The abuse of psychostimulants, such as methamphetamine (METH), is prevalent in young adults and could lead to long-term adaptations in the midbrain dopamine system in abstinent human METH abusers. Nurr1 is a gene that is critical for the survival and maintenance of dopaminergic neurons and has been implicated in dopaminergic neuron related disorders. In this study, we examined the synergistic effects of repeated early exposure to methamphetamine in adolescence and reduction in Nurr1 gene levels. METH binge exposure in adolescence led to greater damage in the nigrostrial dopaminergic system when mice were exposed to METH binge later in life, suggesting a long-term adverse effect on the dopaminergic system. Compared to naïve mice that received METH binge treatment for the first time, mice pretreated with METH in adolescence showed a greater loss of tyrosine hydroxylase (TH) immunoreactivity in striatum, loss of THir fibers in the substantia nigra reticulata (SNr) as well as decreased dopamine transporter (DAT) level and compromised DA clearance in striatum. These effects were further exacerbated in Nurr1 heterozygous mice. Our data suggest that a prolonged adverse effect exists following adolescent METH binge exposure which may lead to greater damage to the dopaminergic system when exposed to repeated METH later in life. Furthermore, our data support that Nurr1 mutations or deficiency could be a potential genetic predisposition which may lead to higher vulnerability in some individuals.     

Multidrug Resistant Acinetobacter baumannii: Risk Factors for Appearance of Imipenem Resistant Strains on Patients Formerly with Susceptible Strains

Background

Multidrug resistant Acinetobacter baumannii (MDRAB) is an important nosocomial pathogen usually susceptible to carbapenems; however, growing number of imipenem resistant MDRAB (IR-MDRAB) poses further clinical challenge. The study was designed to identify the risk factors for appearance of IR-MDRAB on patients formerly with imipenem susceptible MDRAB (IS-MDRAB) and the impact on clinical outcomes.

Methodology/Principal Findings

A retrospective case control study was carried out for 209 consecutive episodes of IS-MDRAB infection or colonization from August 2001 to March 2005. Forty-nine (23.4%) episodes with succeeding clinical isolates of IR-MDRAB were defined as the cases and 160 (76.6%) with all subsequent clinical isolates of IS-MDRAB were defined as the controls. Quantified antimicrobial selective pressure, “time at risk”, severity of illness, comorbidity, and demographic data were incorporated for multivariate analysis, which revealed imipenem or meropenem as the only significant independent risk factor for the appearance of IR-MDRAB (adjusted OR, 1.18; 95% CI, 1.09 to 1.27). With selected cases and controls matched to exclude exogenous source of IR-MDRAB, multivariate analysis still identified carbapenem as the only independent risk factor (adjusted OR, 1.48; 95% CI, 1.14 to 1.92). Case patients had a higher crude mortality rate compared to control patients (57.1% vs. 31.3%, p = 0.001), and the mortality of case patients was associated with shorter duration of “time at risk”, i.e., faster appearance of IR-MDRAB (adjusted OR, 0.9; 95% CI, 0.83 to 0.98).

Conclusions/Significance

Judicious use of carbapenem with deployment of antibiotics stewardship measures is critical for reducing IR-MDRAB and the associated unfavorable outcome.     

Solutions for determining equibiaxial substrate strain for dynamic cell culture

In this work, empirical and analytical solutions of equibiaxial strain on a flexible substrate are derived for a dynamic cell culture system. The empirical formula, which fulfills the mechanistic conditions of the culture system, is based on a regression analysis from finite element analyses for a substrate undergoing large strains (<15%). The analytical (closed-form) solution is derived from the superposition of two elastic responses induced in the equibiaxial strain culture system after applying pressure to a substrate undergoing small strains (microstrains). There is good agreement between the strain predicted from the solutions and from the direct measurement. Using material and geometric properties of the culture system, the solutions developed here are straightforward and can be used to circumvent experimental measurements or finite element analysis to establish substrate pressure–strain relationships.     

The TOR Complex 1 Is Distributed in Endosomes and in Retrograde Vesicles That Form from the Vacuole Membrane and Plays an Important Role in the Vacuole Import and Degradation Pathway

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.