A Highlights from MBoC Selection: Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.     

Oxaliplatin induces immunogenic cells death and enhances therapeutic efficacy of checkpoint inhibitor in a model of murine lung carcinoma

Abstract

Background: Platinum compounds are commonly used for lung cancer treatment. However, the severe side effects and relatively poor prognosis limit their therapeutic effect. Therefore, developing novel platinum derivative and treatment strategy are critical for current lung cancer therapy.

Methods: Flow cytometry, HMGB1 and ATP release, and immunoblotting were performed to evaluate the Oxaliplatin-induced immunogenic cell death (ICD) in two lung carcinoma cells. Vaccination approach and subcutaneous tumor models were created to analyze the tumor regression effect of Oxaliplatin. PD-L1 mRNA and protein levels were detected in LLC (Lewis lung carcinoma). Enhanced therapeutic efficacy of LLC was assessed by co-administration Oxaliplatin and aPD-L1 in murine lung tumor model.

Results: Oxaliplatin induced robust ICD in LLC cells, activated dendritic cells (DCs, CD80⁺CD86⁺) and enhanced cytotoxic T cells (CD8⁺) in LLC tumor tissues, which resulted in tumor regression. Co-administration of Oxaliplatin and checkpoint inhibitor, aPD-L1, could enhance the therapeutic efficacy of LLC in murine lung carcinoma.

Conclusion: This study reveals Oxaliplatin can induce robust ICD in tumor tissues and suppress tumor growth by activating DCs and enhancing T-cell infiltration. Notably, the Oxaliplatin-induced ICD provides an immunogenic microenvironment, which enhances the checkpoint inhibitor therapeutic efficacy of LLC.     

Antiviral Nucleoside Drug Delivery via Amino Acid Phosphoramidates

Abstract

Stable and water soluble amino acid phosphomonoester amidates of AZT were synthesized and shown to have potent anti-HIV-1 activity. Intracellular and cell extract metabolism studies revealed that these compounds are likely to be enzymatically converted to the corresponding monophosphates. In addition, we have shown that the half life and tissue distribution of a phosphoramidate of AZT is 5 and 10-fold greater, respectively, than AZT.     

Influence of community structure on the spatial distribution of critically endangered Dicerandra immaculata var. immaculata (Lamiaceae) at wild, introduced, and extirpated locations in Florida scrub

Community structure at local scales is a major factor controlling population and community dynamics of plant species. Dicerandra immaculata Lakela var. immaculata (Lamiaceae) is a critically endangered plant known only from a few locations in scrub habitat in Florida. Using seven sites where populations of D. immaculata were wild, introduced, and/or extirpated, we sought to answer the following questions: (1) how do habitat characteristics at locations supporting wild D. immaculata plants vary from random locations within the same habitat; (2) how do habitat characteristics differ between wild and extirpated populations; and (3) how do habitat characteristics differ between wild and introduced populations? At locations of wild D. immaculata, community structure had fewer woody stems, shorter understory vegetation, lower percent canopy coverage, and lower percent ground cover of detritus than random locations and locations with extirpated D. immaculata. In addition, bare ground decreased at extirpated locations because other plant species expanded their coverage, water saturation of the soil increased, diversity of shrubs decreased, and composition of the overstory changed compared to that of wild locations. Habitat characteristics associated with introduced plants were more similar to characteristics at randomly chosen locations than those with wild plants. However, introduced plants tended to occupy locations that had drier soil, a higher abundance of conspecifics, and a higher proportion of woody understory plants than that of random locations. Overall, gaps in the canopy and at ground level are likely essential for survival and recruitment of D. immaculata.     

No cumulative effect of 10 years of elevated [CO2] on perennial plant biomass components in the Mojave Desert

Elevated atmospheric CO₂ concentrations ([CO₂]) generally increase primary production of terrestrial ecosystems. Production responses to elevated [CO₂] may be particularly large in deserts, but information on their long‐term response is unknown. We evaluated the cumulative effects of elevated [CO₂] on primary production at the Nevada Desert FACE (free‐air carbon dioxide enrichment) Facility. Aboveground and belowground perennial plant biomass was harvested in an intact Mojave Desert ecosystem at the end of a 10‐year elevated [CO₂] experiment. We measured community standing biomass, biomass allocation, canopy cover, leaf area index (LAI), carbon and nitrogen content, and isotopic composition of plant tissues for five to eight dominant species. We provide the first long‐term results of elevated [CO₂] on biomass components of a desert ecosystem and offer information on understudied Mojave Desert species. In contrast to initial expectations, 10 years of elevated [CO₂] had no significant effect on standing biomass, biomass allocation, canopy cover, and C : N ratios of above‐ and belowground components. However, elevated [CO₂] increased short‐term responses, including leaf water‐use efficiency (WUE) as measured by carbon isotope discrimination and increased plot‐level LAI. Standing biomass, biomass allocation, canopy cover, and C : N ratios of above‐ and belowground pools significantly differed among dominant species, but responses to elevated [CO₂] did not vary among species, photosynthetic pathway (C₃ vs. C₄), or growth form (drought‐deciduous shrub vs. evergreen shrub vs. grass). Thus, even though previous and current results occasionally show increased leaf‐level photosynthetic rates, WUE, LAI, and plant growth under elevated [CO₂] during the 10‐year experiment, most responses were in wet years and did not lead to sustained increases in community biomass. We presume that the lack of sustained biomass responses to elevated [CO₂] is explained by inter‐annual differences in water availability. Therefore, the high frequency of low precipitation years may constrain cumulative biomass responses to elevated [CO₂] in desert environments.     

Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.     

Dickkopf-1 Is Oncogenic and Involved in Invasive Growth in Non Small Cell Lung Cancer

Dickkopf-1 (DKK1) is an inhibitor of the Wnt/β-catenin signaling pathway. However, the role of DKK1 in the progression of non small cell lung cancer (NSCLC) is not fully understood. In this study, RT-PCR and Western blot were used to examine the expression of DKK1 in a panel of ten human NSCLC cell lines and NSCLC tissues. DKK1 expression was highly transactivated in the great majority of these cancer lines. The expression of DKK1 was upregulated on both mRNA and protein levels in NSCLC tissues compared with the adjacent normal lung tissues. Immunohistochemistry and immunofluoresence revealed that DKK1 was mainly distributed in the cytoplasm in both carcinoma tissues and cell lines. DKK1 protein expression was also evaluated in paraffin sections from 102 patients with NSCLC by immunohistochemistry, and 65(63.73%)tumors were DKK1 positive. Relative analysis showed a significant relationship between DKK1 positive expression and lymph node metastasis(P<0.05). Patients with DKK1-positive tumors had poorer DFS than those with negative ESCC (5-year DFS; 15.4% versus 27%, P = 0.007). To further explore the biological effects of DKK1 in NSCLC cells, we over-expressed DKK1 in NSCLC 95C cell using eukaryotic expression vector pCMV-Tab-2b and performed a knockdown of DKK1 in LTEP-a-2 cell using a short hairpin RNA expression vector pSilencer 5.1. DKK1 did not have any effect on proliferation, but seemed to play a role in migration and invasion capability. Overexpression of DKK1 promotes migratory and invasive activity of 95C, while DKK1 knockdown resulted in the suppression of migration and invasion potentials of LTEP-a-2 cell. Taken together, these results indicate that DKK1 may be a crucial regulator in the progression of NSCLC. DKK1 might be a potential therapeutic target in NSCLC.     

miR-homoHSV of Singapore Grouper Iridovirus (SGIV) Inhibits Expression of the SGIV Pro-apoptotic Factor LITAF and Attenuates Cell Death

Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-α (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.