Alterations of proteasome activities in skeletal muscle tissue of diabetic rats

During the last years many investigations have shown that a major catalyst within the mechanism of skeletal muscle wasting occuring under conditions like sepsis, injuries, trauma, cancer cachexia, chronic acidosis, fasting, glucocorticoid treatment, and insulinopenia is the ubiquitin-proteasome system. Evidence for this was obtained by findings that the rate of ATP-dependent protein degradation is increased, that m-RNA concentrations of several proteasome subunits and ubiquitin are increased and the amount of ubiquitin-protein conjugates is elevated under these conditions. Additionally, the enhanced protein breakdown was shown to be suppressed by proteasome inhibitors. In the present report we show that most but not all of the proteolytic activities of partially purified 20S/26S proteasomes from skeletal muscle of rats increase after induction of Diabetes mellitus. This finding suggests that part of the mechanism of acceleration of muscle protein breakdown is due to changes in proteasome activities.     

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC₅₀ for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K_M for l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.     

Germ cell-specific expression of a proacrosin-CAT fusion gene in transgenic mouse testis.

Acrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5' untranslated region of the rat proacrosin gene fused to the CAT-reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10-100 copies per genome in the different strains. The 5' untranslated region of rat proacrosin gene could properly direct CAT-gene expression to spermatocytes and CAT-mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis-acting sequences required for tissue specific proacrosin expression reside on a 2.3-kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.     

Two steps in the intracellular transport of IgD are sensitive to energy depletion

The secretory pathway of murine IgD can be dissected by the use of carbonylcyanide m-chlorophenylhydrazone (CCCP), which inhibits two distinct steps of intracellular transport. The newly synthesized IgD that accumulates at the first step contains high mannose type oligosaccharides which are partially trimmed. The IgD arrested at this step is less processed than the IgD arrested by treatment with monensin. The properties of this biosynthetic intermediate are consistent with inhibition of Ig passage from the endoplasmic reticulum to the Golgi complex. A second CCCP-sensitive step exists in the biosynthesis of IgD, and is characterized by delta-chains that are resistant to endoglycosidase H and contain galactose. This indicates that this second step occurs during or after the passage through the trans-Golgi compartment. The galactose-containing oligosaccharides of the delta-chains arrested at this step do not contain fucose (as do mature, secreted delta-chains). Fucosylation is not inhibited by CCCP, nor is the secretion of fucose-containing delta-chains. These results show that terminal sugars are added to secretory IgD in at least two transport compartments, separable by their sensitivity to CCCP. The inhibition of the secretory pathway at both steps is reversible; upon removal of the drug the arrested IgD is processed normally and is secreted. The sensitivity to CCCP probably reflects transport steps that are sensitive to even partial depletion of ATP, because treatments with other inhibitors of oxidative phosphorylation yield similarly arrested Ig molecules. Thus, by using the protonophore CCCP, we demonstrate two energy-requiring steps in IgD transport which seem to be at two transitions in the secretory pathway. One step is during the passage from the endoplasmic reticulum to the mid-Golgi compartment and the other step is during Ig passage through the trans-Golgi, or subsequent transport to the cell surface.     

Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics*

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.Microglia, along with astrocytes, form the backbone of the immune response in the brain. Microglia, in particular, comprise 10–15% of the brain, varying by region and predominating in areas of the midbrain such as the hippocampus and substantia nigra (1). Separated from the systemic immune system by the blood-brain barrier, the brain''s immune response relies on the ability of microglia to act as a multifaceted immune cell; microglia are able to sense pathogens, toxins, injury, and cytokine levels, as well as respond in a neurotrophic or neurotoxic manner similar to the macrophage in the systemic immune system (2).Microglia can respond to insult and injury in a neurotoxic manner (3, 4) where activated microglia are able to induce pro-inflammatory cytokines to recruit other microglia and astrocytes in response to bacterial infection and produce a wide and varied array of factors including reactive oxygen species (ROS)¹, and reactive nitrogen species (RNS), cytokines and lipid mediators as well as remove cellular debris as a post-infection response through phagocytosis (5). As such, microglia protect themselves from their own toxic products through a series of antioxidant proteins regulated through the actions of nuclear factor, erythroid 2-like 2 protein (NFE2L2) (6). Microglia have been implicated in a growing number of CNS-associated diseases; classically activated microglia have been found in brain regions afflicted with Parkinson''s disease, Alzheimer''s disease, and AIDS-related dementia (7–9). Microglial activation has also been reported to play a role in brain injury because of chronic alcohol exposure (10–13).Raivich et al. described microglia response and phases as a linear set of stages that microglia pass through in response to injury, pathogens, or antibodies from the systemic immune system that have crossed the blood-brain barrier (14). The first stage is a quiescent resting state, followed by an alert stage characterized by increased expression of integrin-binding proteins, or cell adhesion molecules, such as CD11b. The homing stage of activation that follows is characterized by increased cell mobility and adhesion as microglia target sites of injury or invasion. The fourth stage is a phagocytic stage that is often termed the classical microglia response, characterized by production of neurotoxic factors such as ROS through a cell membrane-bound NADPH oxidase complex and RNS through the action of inducible nitric oxide synthase, iNOS, as well as phagocytosis of cellular debris. The final stage, known as the bystander activation stage, potentiates the microglia response by activating additional microglia through the production and release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-6 (IL-6).Our understanding of the role of microglia has broadened in recent years to include neurotrophic as well as neurotoxic features (15, 16). The presence of activated microglia does not always correlate to an inflammatory state in the local brain region, implying a noninflammatory or possibly neurotrophic role for these microglia. Microglia that display multiple activation states have been observed in the brains of Alzheimer''s patients (17). It has been suggested that microglia that enter an inflammatory neurotoxic state first change into a neurotrophic healing response prior to returning to their quiescent resting phase (1). As such, a new schema to describe microglia phenotype was required. M1 phase, which can be triggered in vivo and in vitro by lipopolysaccharide (LPS) and inflammatory cytokines, has been established to describe classically activated microglial cells that are similar to those found in the fourth and fifth stages of Raivich''s microglial hierarchy. Microglia do not return to a resting state without first receiving anti-inflammatory triggers that are released by other microglia. These additional stages have been classified as alternative activation and have multiple healing responses. Microglia can be induced into the first alternative activation stage, M2a, through treatment with interleukin-4 (IL-4), and/or interleukin-13 (IL-13). M2a is a healing phase typified by tissue repair and growth stimulation through the actions of various extracellular matrix factors. Most importantly, M2a microglia act as an anti-inflammatory counterpart to M1 phase microglia by competing for arginine, a nitrogen pool for the production of RNS during M1 phase; M2a phase microglia compete for this pool through the production of arginase-1 (ARG1) which converts arginine into ornithine (18). M2b phase is a mixed activation state that responds to viral infection and activated antibodies characterized by the production of the pro-inflammatory cytokines, TNFα and IL-6, in addition to reduction of IL-12 and increased production of IL-10 (19). M2b phase microglia can be reproduced, in vitro, by treating with IL-1β and LPS concurrently or activated IgA complexes, which bind to Fcγ receptors. M2c phase microglia can be induced through IL-10 exposure in vivo and in vitro, and the emergence of M2c microglia shuts down microglial immune response.In order to study microglia in a laboratory setting, enriched ex vivo microglia, primary microglia, or immortalized cell lines are required. BV2 immortalized mouse microglia have been described as producing 41% of the cytokines and chemokines produced by ex vivo cells as compared with 96% coverage by primary microglia. However, Wilcock et al. showed that BV2 cells were successful at producing the classical activators for all four microglia activation stages as measured by real-time polymerase chain reaction (17). In addition, proteomic analysis of pathway level changes may be able to smooth over the lack of full expression through high levels of accurate protein quantification.Because of their importance in immune response and possible role in multiple disease states, a thorough investigation of the differential proteomic expression in the various microglial activation states is required. Using SILAC-labeled immortalized BV2 microglial cells treated with activators of the various activation stages, a proteome profile that includes the major canonical microglial pathways across all four activation states, providing crucial information as to where in these pathways of various states diverge, was established. In addition, using the differential protein expression data, a novel marker of microglia activation, DAB2, was identified and confirmed in primary mouse microglia through Western blot analysis. The abundance of this protein, as well as other differentially expressed proteins identified in this study, may prove as novel indicators in differentiating and categorizing activated microglia in the brain.     

The cytoskeletal adaptor protein IQGAP1 regulates TCR-mediated signaling and filamentous actin dynamics

The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8(+) T cells. However, IQGAP1-deficient CD8(+) T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.