Person:

Wu, Hao

Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S. cerevisiae Gpi16. We found that the N-terminal caspase-like domain of Gpi8 forms a disulfide-linked dimer, which is strengthened by N-glycosylation. The non-core domain of Gpi8 following the caspase-like domain inhibits this dimerization. In contrast to the previously reported disulfide linkage between Gpi8 and Gpi16 in human and trypanosome GPIT, our data show that the luminal domains of S. cerevisiae Gpi8 and S. cerevisiae Gpi16 do not interact directly, nor do they form a disulfide bond in the intact S. cerevisiae GPIT. Our data suggest that subunit interactions within the GPIT complex from different species may vary, a feature that should be taken into account in future structural and functional studies.

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains.

The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation.

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro–caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASCCARD interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASCPYD filaments for the first time. These data revealed that cross-linking of ASCPYD filaments via ASCCARD mediates the assembly of ASC foci.

Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here, we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of the active DNA demethylation cascade and reveals a regulatory role of 5fC/5caC excision repair in this step-wise process.

We conducted a prospective cohort study among HIV-negative MSM aged 18 years or older between 2007 and 2012 in Beijing, China to measure the rates of incident HIV and identify risk factors for infection. Among 5,800 participants evaluated at enrollment, we identified 486 prevalent cases of HIV (8.4%). Among the 3,625 enrollees who were HIV-negative at enrollment and completed at least one follow-up interview, we identified 440 incident cases of HIV in the follow up period: this constituted an HIV incidence rate of 7.1 per 100 person-years (95% CI: 6.4-7.7). Early treatment of syphilis may have significantly reduced risk of HIV infection (RR: 1.45, 95% CI: 1.11-1.93), while MSM presenting perfect compliance in the cohort did not show reduction in HIV infection. Our study suggested that HIV incidence has been remained high in this sample of Chinese MSM during the intensive preventive intervention, suggesting that we need to find new strategies to prevent HIV infection in this population.

Rett syndrome (RTT) is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2), an epigenetic regulator of mRNA transcription. Here, we report a test of the hypothesis of shared pathophysiology of RTT and fragile X, another monogenic cause of autism and intellectual disability. In fragile X, the loss of the mRNA translational repressor FMRP leads to exaggerated protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5). We found that mGluR5- and protein-synthesis-dependent synaptic plasticity were similarly altered in area CA1 of Mecp2 KO mice. CA1 pyramidal cell-type-specific, genome-wide profiling of ribosome-bound mRNAs was performed in wild-type and Mecp2 KO hippocampal CA1 neurons to reveal the MeCP2-regulated “translatome.” We found significant overlap between ribosome-bound transcripts overexpressed in the Mecp2 KO and FMRP mRNA targets. These tended to encode long genes that were functionally related to either cytoskeleton organization or the development of neuronal connectivity. In the Fmr1 KO mouse, chronic treatment with mGluR5-negative allosteric modulators (NAMs) has been shown to ameliorate many mutant phenotypes by correcting excessive protein synthesis. In Mecp2 KO mice, we found that mGluR5 NAM treatment significantly reduced the level of overexpressed ribosome-associated transcripts, particularly those that were also FMRP targets. Some Rett phenotypes were also ameliorated by treatment, most notably hippocampal cell size and lifespan. Together, these results suggest a potential mechanistic link between MeCP2-mediated transcription regulation and mGluR5/FMRP-mediated protein translation regulation through coregulation of a subset of genes relevant to synaptic functions.

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency. Using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but its drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the fusion oncogene PML-RARα and treats APL in cell and animal models and human patients. ATRA-induced Pin1 ablation also inhibits triple negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.

Atherosclerosis is the primary cause of coronary heart disease (CHD), ischemic stroke, and peripheral arterial disease. Despite effective lipid-lowering therapies and prevention programs, atherosclerosis is still the leading cause of mortality in the United States. Moreover, the prevalence of CHD in developing countries worldwide is rapidly increasing at a rate expected to overtake those of cancer and diabetes. Prominent risk factors include the hardening of arteries and high levels of cholesterol, which lead to the initiation and progression of atherosclerosis. However, cell death and efferocytosis are critical components of both atherosclerotic plaque progression and regression, yet, few currently available therapies focus on these processes. Thus, understanding the causes of cell death within the atherosclerotic plaque, the consequences of cell death, and the mechanisms of apoptotic cell clearance may enable the development of new therapies to treat cardiovascular disease. Here, we review how endoplasmic reticulum stress and cholesterol metabolism lead to cell death and inflammation, how dying cells affect plaque progression, and how autophagy and the clearance of dead cells ameliorates the inflammatory environment of the plaque. In addition, we review current research aimed at alleviating these processes and specifically targeting therapeutics to the site of the plaque.

Inflammasomes are cytosolic caspase-1 activation complexes that sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic interactions by Pyrin domains (PYD) and caspase recruitment domains (CARD) in inflammasome component proteins mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only the interaction domains exert inhibitory effects on inflammasome assembly. In this study, we determined the structure of human caspase-1CARD filament by cryo-electron microscopy and investigated the biophysical properties of two caspase-1-like CARD-only proteins, human inhibitor of CARD (INCA or CARD17) and ICEBERG (or CARD18). Our results reveal the surprising finding that INCA caps caspase-1 filament, thereby exerting potent inhibition with low nanomolar Ki on caspase-1CARD polymerization in vitro and inflammasome activation in cells. While caspase-1CARD uses six complementary surfaces of three types for filament assembly, INCA is defective in two of the six interfaces to terminate caspase-1 filament.