Walter J. Esselman

Identification of APOBEC3DE as Another Antiretroviral Factor from the Human APOBEC Family

ABSTRACT A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.

Inhibition of PTPs by H2O2 regulates the activation of distinct MAPK pathways

It has been shown that endogenous production of reactive oxygen species (ROS) during T cell activation regulates signaling events including MAPK activation. Protein tyrosine phosphatases (PTPs) have been regarded as targets of ROS which modify the catalytic cysteine residues of the enzymes. We have analyzed the interplay between the inhibition of PTPs and the activation of MAPK by H(2)O(2). Stimulation of Jurkat T cells with H(2)O(2) induces the phosphorylation of ERK, p38, and JNK members of MAPK family. H(2)O(2) stimulation of T cells was found to inhibit the PTP activity of CD45, SHP-1, and HePTP. Transfection of cells with wtSHP-1 decreased H(2)O(2)-induced ERK and JNK phosphorylation without affecting p38 phosphorylation. Transfection with wtHePTP inhibited H(2)O(2)-induced ERK and p38 phosphorylation without inhibiting JNK phosphorylation. The Src-family kinase inhibitor, PP2, inhibited the H(2)O(2)-induced phosphorylation of ERK, p38, and JNK. The phospholipase C (PLC) inhibitor, U73122, or the protein kinase C (PKC) inhibitor, Ro-31-8425, blocked H(2)O(2)-induced ERK phosphorylation, whereas the same treatment did not inhibit p38 or JNK phosphorylation. Taken together, these results suggest that inhibition of PTPs by H(2)O(2) contributes to the induction of distinct MAPK activation profiles via differential signaling pathways.

Diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock

The present epidemic of diabetes is resulting in a worldwide increase in cardiovascular and microvascular complications including retinopathy. Current thinking has focused on local influences in the retina as being responsible for development of this diabetic complication. However, the contribution of circulating cells in maintenance, repair, and dysfunction of the vasculature is now becoming appreciated. Diabetic individuals have fewer endothelial progenitor cells (EPCs) in their circulation and these cells have diminished migratory potential, which contributes to their decreased reparative capacity. Using a rat model of type 2 diabetes, we show that the decrease in EPC release from diabetic bone marrow is caused by bone marrow neuropathy and that these changes precede the development of diabetic retinopathy. In rats that had diabetes for 4 mo, we observed a dramatic reduction in the number of nerve terminal endings in the bone marrow. Denervation was accompanied by increased numbers of EPCs within the bone marrow but decreased numbers in circulation. Furthermore, denervation was accompanied by a loss of circadian release of EPCs and a marked reduction in clock gene expression in the retina and in EPCs themselves. This reduction in the circadian peak of EPC release led to diminished reparative capacity, resulting in the development of the hallmark feature of diabetic retinopathy, acellular retinal capillaries. Thus, for the first time, diabetic retinopathy is related to neuropathy of the bone marrow. This novel finding shows that bone marrow denervation represents a new therapeutic target for treatment of diabetic vascular complications.

The Unconventional Role of Acid Sphingomyelinase in Regulation of Retinal Microangiopathy in Diabetic Human and Animal Models

OBJECTIVE Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study. RESEARCH DESIGN AND METHODS Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies. RESULTS We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α–induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM−/− mice or inhibition of ASM activity by DHA prevents acellular capillary formation. CONCLUSIONS This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.

Inhibition of cytokine signaling in human retinal endothelial cells through downregulation of sphingomyelinases by docosahexaenoic acid.

PURPOSE The authors have previously demonstrated that DHA inhibits cytokine-induced inflammation in human retinal endothelial cells (HRECs), the resident vasculature affected by diabetic retinopathy. However, the anti-inflammatory mechanism of docosahexaenoic acid (DHA) is still not well understood. Sphingolipids represent a major component of membrane microdomains, and ceramide-enriched microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) are key regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis to proinflammatory ceramide. The authors address the hypothesis that DHA inhibits cytokine-induced inflammatory signaling in HRECs by downregulating sphingomyelinases. METHODS ASMase and NSMase activity was determined by sphingomyelinase assay in primary cultures of HRECs. The expression of ASMase, NSMase, ICAM-1, and VCAM-1 was assessed by quantitative PCR and Western blot analysis. Gene silencing of ASMase and NSMase was obtained by siRNA treatment. RESULTS Inflammatory cytokines TNFalpha and IL-1beta induced cellular adhesion molecule (CAM) expression and rapid increase in ASMase and NSMase activity in HRECs. DHA decreased basal and cytokine-induced ASMase and NSMase expression and activity and the upregulation of CAM expression. Anti-inflammatory effects of DHA on cytokine-induced CAM expression were mimicked by inhibition/gene silencing of ASMase and NSMase. The sphingomyelinase pathway rather than ceramide de novo synthesis pathway was important for inflammatory signaling in HRECs. CONCLUSIONS This study provides a novel potential mechanism for the anti-inflammatory effect of DHA in HRECs. DHA downregulates the basal and cytokine-induced ASMase and NSMase expression and activity level in HRECs, and inhibition of sphingomyelinases in endothelial cells prevents cytokine-induced inflammatory response.

PHOSPHORYLATION OF CD45 BY CASEIN KINASE 2 : MODULATION OF ACTIVITY AND MUTATIONAL ANALYSIS

CD45 is a receptor-type protein-tyrosine phosphatase (PTP) that is required for antigen-specific stimulation and proliferation in lymphocytes. This study was designed to determine the nature of specific kinases in lymphocytes that phosphorylate CD45 and to determine the effect of phosphorylation on CD45 PTP activity. A major cytoplasmic lymphocyte kinase that phosphorylated CD45 was identified as casein kinase 2 (CK2) by use of an in-gel kinase assay in combination with immunoprecipitation, immunodepletion, and specific inhibition. Mutational analysis of CK2 consensus sites showed that the target for CK2 was in an acidic insert of 19 amino acids in the D2 domain, and Ser to Ala mutations at amino acids 965, 968, 969, and 973 abrogated CK2 phosphorylation of CD45. CK2 phosphorylation increased CD45 activity 3-fold toward phosphorylated myelin basic protein, and this increase was reversible by PP2A treatment. Mutation of Ser to Glu at the CK2 sites had the same effect as phosphorylation and also tripled the V max of CD45. CD45 isolatedin vivo was highly phosphorylated and could not be phosphorylated by CK2 without prior dephosphorylation with phosphatase PP2A. We conclude that CK2 is a major lymphocyte kinase that is responsible for in vivo phosphorylation of CD45, and phosphorylation at specific CK2 sites regulates CD45 PTP activity.

Substrate Conformational Restriction and CD45-catalyzed Dephosphorylation of Tail Tyrosine-phosphorylated Src Protein

Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83–533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565–1268), we have analyzed the kinetic behavior of dephosphorylation. A time course of phosphatase activity showed the presence of a burst phase. By varying the concentration of P-Src, it was shown that the amplitude of this burst phase increased linearly with respect to P-Src concentration. Approximately 2% of P-Src was shown to be rapidly dephosphorylated followed by a slower linear phase. A P-Src protein substrate containing a functional point mutation in the Src homology domain 2 (SH2) led to more rapid dephosphorylation catalyzed by CD45, and this reaction showed only a single linear kinetic phase. These results were interpreted in terms of a model in which P-Src exists in a relatively slow dynamic equilibrium between “closed” and “open” conformational forms. Combined mutations in the SH2 and SH3 domain or the addition of an SH3 domain ligand peptide enhanced the accessibility of P-Src to CD45 by biasing P-Src to a more open form. Consistent with this model, a phosphotyrosine peptide that behaved as an SH2 domain binding ligand showed ∼100-fold greater affinity for unphosphorylated Srcversus P-Src. Surprisingly, P-Src possessing combined SH3 and SH2 functional inactivating point mutations was dephosphorylated by CD45 more slowly compared with P-Src completely lacking SH3 and SH2 domains. Additional data suggest that the SH3 and SH2 domains can inhibit accessibility of the P-Src tail to CD45 by interactions other than direct phosphotyrosine binding by the SH2 domain. Taken together, these results suggest how activation of Src family member signaling pathways by CD45 may be influenced by the presence or absence of ligand interactions remote from the tail.

The isolation and specificity of alfalfa lipoxygenase

Partial purification of the lipoxygenase from alfalfa seed was accomplished by fractionation of the protein with (NH4)2SO4, phosphate, heavy metal salts and ultracentrifugation. About 24% of the original activity was recovered. The partially-purified alfalfa lipoxygenase enzyme was free of hydroperoxide-decomposing activity and was used to determine the positional specificity of linoleic acid oxidation by alfalfa lipoxygenase. Combined gas liquid chromatography-mass spectrometry was used to analyze known mixtures of 10- and 12-hydroxystearic acid derivatives and was satisfactory for the quantitative determination of the ratio of each component. This combination was used to analyze mixtures containing position isomers of hydroxy fatty acids without separation of each individual compound by other methods. Hydroperoxides produced from linoleic acid oxidation catalyzed by alfalfa lipoxygenase were converted by sodium borohydride reduction, catalytic hydrogenation and bis(trimethylsilyl)acetamide silylation to their corresponding trimethylsilyl either esters and the positional distribution was studied. The 9- and 13-linoleate hydroperoxides produced by alfalfa lipoxygenase were in equal concentrations (50∶50) whereas the distribution for soybean lipoxygenase was 70% 13- and 30% 9-hydroperoxides.

Examining the role of lipid mediators in diabetic retinopathy.

Abstract Diabetic retinopathy is the most disabling complication of diabetes, affecting 65% of patients after 10 years of the disease. Current treatment options for diabetic retinopathy are highly invasive and fall short of complete amelioration of the disease. Understanding the pathogenesis of diabetic retinopathy is critical to the development of more effective treatment options. Diabetic hyperglycemia and dyslipidemia are the main metabolic insults that affect retinal degeneration in diabetes. Although the role of hyperglycemia in inducing diabetic retinopathy has been studied in detail, much less attention has been paid to dyslipidemia. Recent clinical studies have demonstrated a strong association between dyslipidemia and development of diabetic retinopathy, highlighting the importance of understanding the exact changes in retinal lipid metabolism in diabetes. This review describes what is known on the role of dyslipidemia in the development of diabetic retinopathy, with a focus on retinal-specific lipid metabolism and its dysregulation in diabetes.

cAMP potentiates H2O2-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation

In Jurkat T lymphocytes, hydrogen peroxide (H(2)O(2)) potentiates the phosphorylation level of extracellular signal-regulated kinase 1 and 2 (ERK1/2) caused by T cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 or anti-CD3 alone. Submillimolar concentrations of H(2)O(2)-induced phosphorylation of ERK1/2 and MAP/ERK kinase 1 and 2 (MEK1/2) without antigenic stimulation. H(2)O(2) also induced the electrophoretic mobility shift of Lck from 56 to 60 kDa. The MEK inhibitor, PD98059 attenuated ERK1/2 and MEK1/2 phosphorylation, as well as the migration shift of Lck induced by H(2)O(2). The phospholipase C (PLC) inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and MEK1/2 induced by H(2)O(2). Interestingly, an increase of intracellular cAMP level with forskolin or 8-(4-chlorophenylthio)-cAMP augmented ERK1/2 phosphorylation by H(2)O(2), while inhibiting MEK1/2 phosphorylation by H(2)O(2). These results demonstrate an alternative pathway that results in augmentation of ERK1/2 phosphorylation without concomitant MEK1/2 phosphorylation in T cells.