Ashley L. McCormack

Quantitative Membrane Proteomics Reveals New Cellular Targets of Viral Immune Modulators

Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma–associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif–deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.

The Hydrogen Peroxide Reactivity of Peptidylglycine Monooxygenase Supports a Cu(II)-Superoxo Catalytic Intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H2O2, the α-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When 18O-labeled H2O2 was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with 18O and must therefore be derived from H2O2. However, when the reaction was carried out with H 162O2 in the presence of 18O2, 60% of the product contained the 18O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the CuH center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.

Quantitative proteomics identifies a change in glial glutamate metabolism at the time of female puberty

Mammalian puberty requires activation of luteinizing hormone-releasing hormone (LHRH) neurons. In turn, these neurons are controlled by transsynaptic and glia-to-neuron communication pathways, which employ diverse cellular proteins for proper function. We have now used a high throughput relative quantitative proteomics technique to identify such proteins. We selected the method of two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) and cleavable isotope-coded affinity tags (cICAT), to both identify and quantify individual proteins within a complex protein mixture. The proteins used derived from the hypothalamus of juvenile (25-day-old) and peripubertal (first proestrus, LP) female rats, and their identity was established by analyzing their mass spectra via database searching. Five proteins involved in glutamate metabolism were detected and two of them appeared to be differentially expressed. They were selected for further analysis, because of their importance in controlling glutamate synthesis and degradation, and their preferential expression in astroglial cells. One, glutamate dehydrogenase (GDH) catalyzes glutamate synthesis; its hypothalamic content detected by 2DLC-MS/MS increases at first proestrus. The other, glutamine synthetase (GS), catalyzes the metabolism of glutamate to glutamine; its content decreases in proestrus. Western blot analysis verified these results. Because these changes suggested an increased glutamate production at puberty, we measured glutamate release from hypothalamic fragments from juvenile 29-day old rats, and from rats treated with PMSG to induce a premature proestrus surge of luteinizing hormone (LH). To determine the net output of glutamate in the absence of re-uptake we used the excitatory amino acid transporter (EAAT) inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). PDC elicited significantly more glutamate- and LHRH-release from the proestrus hypothalamus. Thus, an increase excitatory drive to the LHRH neuronal network provided by glutamatergic inputs of glial origin, is an event contributing to the pubertal activation of LHRH secretion.

Remodeling of protein and mRNA expression in Leishmania mexicana induced by deletion of glucose transporter genes.

Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of Leishmania mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose.