Par-4 Links Dopamine Signaling and Depression

SummaryProstate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the calmodulin binding motif in the third cytoplasmic loop. Calmodulin can effectively compete with Par-4 binding in a Ca2+-dependent manner, providing a route for Ca2+-mediated downregulation of D2DR efficacy. To examine the importance of the Par-4/D2DR interaction in dopamine signaling in vivo, we used a mutant mouse lacking the D2DR interaction domain of Par-4, Par-4ΔLZ. Primary neurons from Par-4ΔLZ embryos exhibit an enhanced dopamine-cAMP-CREB signaling pathway, indicating an impairment in dopamine signaling in these cells. Remarkably, Par-4ΔLZ mice display significantly increased depression-like behaviors. Collectively, these results provide evidence that Par-4 constitutes a molecular link between impaired dopamine signaling and depression

Persistence of Different Forms of Transient RNAi during Apoptosis in Mammalian Cells

Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2–3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events

Regulation of activation-induced deaminase stability and antibody gene diversification by Hsp90

Hsp90 stabilizes and prevents degradation of cytoplasmic activation-induced deaminase

Targeting of AMSH to endosomes is required for epidermal growth factor receptor degradation

To reach the lysosomes, down-regulated receptors such as the epidermal growth factor receptor must first be sorted into internal vesicles of late endosomes (multivesicular bodies), a ubiquitin-dependent event that requires the coordinated function of the endosome sorting complex required for transport (ESCRT) proteins. Here we report that CHMP3, an ESCRT-III complex component, and associated molecule of SH3 domain of STAM (AMSH), a deubiquitinating enzyme, interact with each other in cells. A dominant-negative version of CHMP3, which specifically prevents targeting of AMSH to endosomes, inhibits degradation but not internalization of EGFR, suggesting that endosomal AMSH is a functional component of the multivesicular body pathway

Electron transfer dissociation (ETD): The mass spectrometric breakthrough essential for O‐GlcNAc protein site assignments—a study of the O‐GlcNAcylated protein Host Cell Factor C1

The development of electron-based, unimolecular dissociation MS, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile PTM site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD MS. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation MS, in a study of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional coregulator that forms a stable complex with O-GlcNAc transferase and controls cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) MS, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include 19 sites of O-GlcNAcylation, two sites of phosphorylation, and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation

Ikaros and GATA-1 Combinatorial Effect Is Required for Silencing of Human γ-Globin Genes▿

During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human β-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human γ-gene promoters, thereby contributing to γ-globin gene silencing at the time of the γ- to β-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human γ-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation

Essential Dosage-Dependent Functions of the Transcription Factor Yin Yang 1 in Late Embryonic Development and Cell Cycle Progression

Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing ∼75%, ∼50%, and ∼25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations