Multiple APOBEC3 restriction factors for HIV-1 and one Vif to rule them all.

Several members of the APOBEC3 family of cellular restriction factors provide intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection against HIV-1Δvif through hypermutation of the viral genome, inhibition of reverse transcription, and inhibition of viral DNA integration into the host genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif, which contains distinct domains that specifically interact with these APOBEC3 proteins to ensure their proteasomal degradation, allowing virus replication to proceed. Here, we review our current understanding of APOBEC3 structure, editing and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3 interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3 proteins to restriction and control of HIV-1 replication in infected patients.

Cardiomyocyte apoptosis in heart development: methods and protocols.

Apoptosis is the process of programmed cell death that has been identified in the development of heart. It is likely to be regulated by survival and death signals that are also present in many other tissues. To understand cardiomyocyte apoptosis in normal and abnormal development of heart, the cell death detection techniques were applied to various researches. These methods include morphological, histological, or molecular assays based on recent advances in our understanding of the molecular mechanism of cell death, including: (1) terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling of fragmented nuclei, (2) cardiovascular molecular imaging of apoptosis using Annexin V, and (3) immunohistochemical detection of activated caspases.

The putative tumor suppressor Zc3h12d modulates toll-like receptor signaling in macrophages.

Toll-like receptors (TLR) are pivotal in macrophage activation. The molecular mechanisms controlling TLR signaling and macrophage activation are not completely understood. Zc3h12d is originally identified as a possible tumor suppressor gene. However, its function remains unknown. We here report that Zc3h12d negatively regulates TLR signaling and macrophage activation. Zc3h12d was enriched in spleen, lung and lymph node. In macrophages, the expression of Zc3h12d was remarkably induced by TLR ligands through JNK and NF-κB signal pathways. On the other hand, overexpression of Zc3h12d significantly inhibited TLR2 and TLR4 activation-induced JNK, ERK and NF-κB signaling as well as macrophage inflammation. Similar to Zc3h12a/MCPIP1, Zc3h12d also decreased the global cellular protein ubiquitination. These findings suggest that Zc3h12d is a novel negative feedback regulator of TLR signaling and macrophage activation and thus may play a role in host immunity and inflammatory diseases.

Monocyte chemotactic protein-induced protein 1 (MCPIP1) suppresses stress granule formation and determines apoptosis under stress.

It is unclear how stress granule (SG) formation and cellular apoptosis are coordinately regulated. MCPIP1 (monocyte chemotactic protein-induced protein 1), also known as Zc3h12a, is a critical regulator of the inflammatory response and immune homeostasis. However, the role of MCPIP1 in stress response remains unknown. Here, we report that overexpression of MCPIP1 inhibited the assembly of SGs in response to various stresses. Conversely, MCPIP1-deficient splenocytes developed more SGs even without stress. On the other hand, overexpression of MCPIP1 sensitized RAW 264.7 cells to apoptosis under stress, whereas MCPIP1-deficient cells were resistant to stress-induced apoptosis. Mutagenesis study showed that the ability of MCPIP1 to repress SG formation is dependent on its deubiquitinating activity. Consistently, MCPIP1 negatively regulated stress-induced phosphorylation of eIF2α and thus released stress-induced inhibition of protein translation. However, MCPIP1 also inhibited 15-deoxy-Δ(12,14)-prostaglandin J(2)-induced SG formation, which was reported to be independent of eIF2α phosphorylation. Taken together, these results suggest that MCPIP1 coordinates SG formation and apoptosis during cellular stress and may play a critical role in immune homeostasis and resolution of macrophage inflammation.

MCP-1 upregulates amylin expression in murine pancreatic ß cells through ERK/JNK-AP1 and NF-κB related signaling pathways independent of CCR2.

BACKGROUND: Amylin is the most abundant component of islet amyloid implicated in the development of type 2 diabetes. Plasma amylin levels are elevated in individuals with obesity and insulin resistance. Monocyte chemoattractant protein-1 (MCP-1, CCL2) is involved in insulin resistance of obesity and type 2 diabetes. We investigated the effect of MCP-1 on amylin expression and the underlying mechanisms with murine pancreatic ß-cell line MIN6 and pancreatic islets. METHODOLOGY/PRINCIPAL FINDINGS: We found that MCP-1 induced amylin expression at transcriptional level and increased proamylin and intermediate forms of amylin at protein level in MIN6 cells and islets. However, MCP-1 had no effect on the expressions of proinsulin 1 and 2, as well as prohormone convertase (PC) 1/3 and PC2, suggesting that MCP-1 specifically induces amylin expression in ß-cells. Mechanistic studies showed that although there is no detectable CCR2 mRNA in MIN6 cells and islets, pretreatment of MIN6 cells with pertussis toxin inhibited MCP-1 induced amylin expression, suggesting that alternative Gi-coupled receptor(s) mediates the inductive effect of MCP-1. MCP-1 rapidly induced ERK1/2 and JNK phosphorylation. Inhibitors for MEK1/2 (PD98059), JNK (SP600125) or AP1 (curcumin) significantly inhibited MCP-1-induced amylin mRNA expression. MCP-1 failed to induce amylin expression in pancreatic islets isolated from Fos knockout mice. EMSA showed that JNK and ERK1/2 were involved in MCP-1-induced AP1 activation. These results suggest that MCP-1 induces murine amylin expression through AP1 activation mediated by ERK1/2 or JNK. Further studies showed that treatment of MIN6 cells with NF-κB inhibitor or overexpression of IκBα dominant-negative construct in MIN6 cells significantly inhibited MCP-1-induced amylin expression, suggesting that NF-κB related signaling also participates in MCP-1-induced murine amylin expression. CONCLUSIONS/SIGNIFICANCE: MCP-1 induces amylin expression through ERK1/2/JNK-AP1 and NF-κB related signaling pathways independent of CCR2. Amylin upregulation by MCP-1 may contribute to elevation of plasma amylin in obesity and insulin resistance.

MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-kappaB signaling.

The intensity and duration of macrophage-mediated inflammatory responses are controlled by proteins that modulate inflammatory signaling pathways. MCPIP1 (monocyte chemotactic protein-induced protein 1), a recently identified CCCH Zn finger-containing protein, plays an essential role in controlling macrophage-mediated inflammatory responses. However, its mechanism of action is poorly understood. In this study, we show that MCPIP1 negatively regulates c-Jun N-terminal kinase (JNK) and NF-κB activity by removing ubiquitin moieties from proteins, including TRAF2, TRAF3, and TRAF6. MCPIP1-deficient mice spontaneously developed fatal inflammatory syndrome. Macrophages and splenocytes from MCPIP1(-/-) mice showed elevated expression of inflammatory gene expression, increased JNK and IκB kinase activation, and increased polyubiquitination of TNF receptor-associated factors. In vitro assays directly demonstrated the deubiquitinating activity of purified MCPIP1. Sequence analysis together with serial mutagenesis defined a deubiquitinating enzyme domain and a ubiquitin association domain in MCPIP1. Our results indicate that MCPIP1 is a critical modulator of inflammatory signaling.

Fatty acids induce amylin expression and secretion by pancreatic beta-cells.

Amylin is the major component of pancreatic amyloid, which is implicated in the development of type 2 diabetes. It is costored with insulin in the secretory granules of pancreatic beta-cells and cosecreted with insulin following stimulation with glucose. Here, we investigate the effect of fatty acids (FAs) on amylin expression and secretion by beta-cells and explore the underlying mechanisms. Palmitate and oleate dose-dependently induced amylin mRNA accumulation in murine pancreatic beta-cell line MIN6 and primary pancreatic islets. the inductive effect of FAs on amylin expression is independent of glucose concentration. FAs upregulated amylin expression at the transcriptional level, and FAs must be metabolized to induce amylin expression. FAs also significantly induced human amylin promoter activation. Pretreatment of MIN6 cells with Ca(2+) chelator (EGTA, BAPTA-AM) PKC inhibitor Gö-6976 or protein synthesis inhibitor cycloheximide significantly inhibited FA-induced amylin mRNA expression. Transcription factors cAMP-responsive element-binding protein, pancreatic and duodenal homeobox factor-1, and peroxisome proliferator-activated receptor were not involved in FA-induced amylin expression. Palmitate and oleate both increased amylin and insulin release from MIN6 cells and stimulated amylin expression but had no effect on insulin expression. Mice refed with Intralipid had significantly higher levels of plasma FFA, amylin, and insulin than those refed with saline. These data demonstrate that FAs differently regulate amylin and insulin expression and induce both amylin and insulin release. Ca(2+) and PKC signaling pathways and de novo-synthesized protein(s) were involved in FA-induced amylin expression. Induction of amylin production and release by FA may contribute to its biological functions under physiological conditions.

Mechanisms of glucose-induced expression of pancreatic-derived factor in pancreatic beta-cells.

Pancreatic-derived factor (PANDER) is a cytokine-like peptide highly expressed in pancreatic beta-cells. PANDER was reported to promote apoptosis of pancreatic beta-cells and secrete in response to glucose. Here we explored the effects of glucose on PANDER expression, and the underlying mechanisms in murine pancreatic beta-cell line MIN6 and primary islets. Our results showed that glucose up-regulated PANDER mRNA and protein levels in a time- and dose-dependent manner in MIN6 cells and pancreatic islets. In cells expressing cAMP response element-binding protein (CREB) dominant-negative construct, glucose failed to induce PANDER gene expression and promoter activation. Treatment of the cells with calcium chelator [EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM)], the voltage-dependent Ca(2+) channel inhibitor (nifedipine), the protein kinase A (PKA) inhibitor (H89), the protein kinase C (PKC) inhibitor (Go6976), or the MAPK kinase 1/2 inhibitor (PD98059), all significantly inhibited glucose-induced PANDER gene expression and promoter activation. Further studies showed that glucose induced CREB phosphorylation through Ca(2+)-PKA-ERK1/2 and Ca(2+)-PKC pathways. Thus, the Ca(2+)-PKA-ERK1/2-CREB and Ca(2+)-PKC-CREB signaling pathways are involved in glucose-induced PANDER gene expression. Wortmannin (phosphatidylinositol 3-kinase inhibitor), ammonium pyrrolidinedithiocarbamate (nuclear factor-kappaB inhibitor and nonspecific antioxidant), and N-acetylcysteine (antioxidant) were also found to inhibit glucose-induced PANDER promoter activation and gene expression. Because there is no nuclear factor-kappaB binding site in the promoter region of PANDER gene, these results suggest that phosphatidylinositol 3-kinase and reactive oxygen species be involved in glucose-induced PANDER gene expression. In conclusion, glucose induces PANDER gene expression in pancreatic beta-cells through multiple signaling pathways. Because PANDER is expressed by pancreatic beta-cells and in response to glucose in a similar way to those of insulin, PANDER may be involved in glucose homeostasis.

The immunopharmacological properties of transforming growth factor beta.

Transforming growth factor-beta (TGF-beta) family members are multifunctional molecules, which play pivotal roles in regulating cell proliferation, differentiation, migration, development, tissue remodeling and repair. These events are closely associated with host immune responses and inflammation. Despite some controversies on their function in controlling dendritic and T regulatory cell development and activity, the importance of TGF-betas in the progress of autoimmunity and inflammatory diseases has been well appreciated and new aspects of their contribution continue to be recognized. Since one of the major biological properties of TGF-betas is its capacity to potently suppress immune responses, they are considered as candidates for the development of therapeutic agents to fend off undesirable damage associated with immune and inflammatory conditions.