Ray Lu

Interaction of the porcine reproductive and respiratory syndrome virus nucleocapsid protein with the inhibitor of MyoD family-a domain-containing protein.

Abstract Porcine reproductive and respiratory syndrome (PRRS) virus is an RNA virus that replicates in the cytoplasm, but the viral nucleocapsid (N) protein localizes specifically in the nucleus and nucleolus of virus-infected cells. Nuclear localization of N is non-essential for PRRSV replication in cultured cells but has been shown to modulate the pathogenesis of virus in pigs, suggesting that N plays an accessory role in the nucleus during infection. We identified by yeast two-hybrid screening the inhibitor of MyoD family-a (I-mfa) domain-containing protein (HIC) as a cellular partner for PRRS virus (PRRSV) N protein. This protein is a homolog of human HIC, a recently identified cellular transcription factor. The specific interaction of PRRSV N with HIC was confirmed in cells by mammalian two-hybrid assay and co-immunoprecipitation and in vitro by GST pull-down assay. HIC is a zinc-binding protein and confocal microscopy demonstrated co-localization of N with the HIC-p40 isomer in the nucleus and nucleolus, and in the cytoplasm with HIC-p32, which is the N-terminal truncation of HIC-p40. The porcine homolog of HIC is universally expressed in pig tissues including alveolar macrophages. The interaction of viral capsid with the cellular transcription factor implicates a possible regulation of host cell gene expression by the N protein during PRRSV infection.

Luman/CREB3 Recruitment Factor Regulates Glucocorticoid Receptor Activity and Is Essential for Prolactin-Mediated Maternal Instinct

ABSTRACT The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system in animal responses to stress. It is known that the HPA axis is attenuated at parturition to prevent detrimental effects of glucocorticoid secretion including inhibition of lactation and maternal responsiveness. Luman/CREB3 recruitment factor (LRF) was identified as a negative regulator of CREB3 which is involved in the endoplasmic reticulum stress response. Here, we report a LRF gene knockout mouse line that has a severe maternal behavioral defect. LRF−/− females lacked the instinct to tend pups; 80% of their litters died within 24 h, while most pups survived if cross-fostered. Prolactin levels were significantly repressed in lactating LRF−/− dams, with glucocorticoid receptor (GR) signaling markedly augmented. In cell culture, LRF repressed transcriptional activity of GR and promoted its protein degradation. LRF was found to colocalize with the known GR repressor, RIP140/NRIP1, which inhibits the activity by GR within specific nuclear punctates that are similar to LRF nuclear bodies. Furthermore, administration of prolactin or the GR antagonist RU486 restored maternal responses in mutant females. We thus postulate that LRF plays a critical role in the attenuation of the HPA axis through repression of glucocorticoid stress signaling during parturition and the postpartum period.

JAB1/CSN5 inhibits the activity of Luman/CREB3 by promoting its degradation

Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound transcription factor that has been implicated in the ER stress response. In this study, we used the region of Luman containing the basic DNA-binding domain as bait in a yeast two-hybrid screen and identified the Jun activation domain-binding protein 1 (JAB1) or the COP9 signalosome complex unit 5 (CSN5) as an interacting protein. We confirmed their direct binding by glutathione S-transferase pull-down assays, and verified the existence of such interaction in the cellular environment by mammalian two-hybrid and co-immunoprecipitation assays. Deletion mapping studies revealed that the MPN domain in JAB1 was essential and sufficient for the binding. JAB1 also colocalized with Luman in transfected cells. More interestingly, the nuclear form of Luman was shown to promote the translocation of JAB1 into the nucleus. We found that overexpression of JAB1 shortened the half-life of Luman by 67%, and repressed its transactivation function on GAL4 and unfolded protein response element (UPRE)-containing promoters. We therefore propose that JAB1 is a novel binding partner of Luman, which negatively regulates the activity of Luman by promoting its degradation.

LUMAN/CREB3 is a key regulator of glucocorticoid-mediated stress responses.

Altered glucocorticoid sensitivity is believed to contribute to a number of human diseases, including inflammatory and autoimmune conditions as well as disorders characterized by abnormal hypothalamic-pituitary-adrenal axis (HPA) function. LUMAN (or CREB3), originally identified through its interaction with a cell cycle regulator HCFC1, is an endoplasmic reticulum membrane-bound transcription factor that is involved in the unfolded protein response. Here we demonstrate that LUMAN changes the glucocorticoid response by modulating the expression of the glucocorticoid receptor leading to an overall increase in GR activity. Luman-deficient mice exhibited a blunted stress response characterized by low levels of both anxiety and depressive-like behaviour in addition to low circulating corticosterone levels. These mice also have reduced dendritic branching in the CA3 region of the hippocampus, consistent with increased GR responses. These findings are consistent with the notion that elevated GR activities are the primary cause of the observed phenotype in these LUMAN-deficient mice. We thus postulate that LUMAN is a key regulator of GR-mediated signaling and modulates HPA axis reactivity.

LUMAN/CREB3 Plays a Dual Role in Stress Responses as a Cofactor of the Glucocorticoid Receptor and a Regulator of Secretion

LUMAN/CREB3, originally identified through its interaction with a cell cycle regulator HCFC1, is a transcription factor involved in the unfolded protein response during endoplasmic reticulum stress. Previously using gene knockout mouse models, we have shown that LUMAN modulates the glucocorticoid (GC) response leading to enhanced glucocorticoid receptor (GR) activity and lower circulating GC levels. Consequently, the stress response is dysregulated, leading to a blunted stress response in the Luman-deficient mice. One question that remained was how LUMAN deficiency affected the stress response at the cellular level leading to the changes in the physiological stress response. Here, we found that LUMAN interacts with GR through a putative nuclear receptor box site and can activate GR in the absence of a ligand. Further investigation showed that, when activated, LUMAN binds to the glucocorticoid response element (GRE), increasing the activity of GR exponentially compared to GR-ligand binding alone. On the other hand, we also found that in the absence of LUMAN, cells were more sensitive to cellular stress, exhibiting decreased secretory capacity. Hence our current data suggest that LUMAN may function both as a transcriptional cofactor of GR and a hormone secretion regulator, and through this, plays a role in stress sensitivity and reactivity to stress.

Characterization of nuclear foci-targeting of Luman/CREB3 recruitment factor (LRF/CREBRF) and its potential role in inhibition of herpes simplex virus-1 replication

The recently identified Luman/CREB3-binding partner LRF (Luman/CREB3 recruitment factor) was shown to localize to discrete sub-nuclear foci. Luman is implicated in herpes simplex virus-1 (HSV-1) latency/reactivation and the unfolded protein response (UPR) pathway; therefore, we sought to characterize the formation of the LRF nuclear foci in the context of cellular signaling and HSV-1 replication. Here, we mapped the nuclear foci-targeting sequence to the central region containing the first leucine zipper (a.a.415-519), and found that the integrity of the whole region appears essential for LRF foci formation. LRF foci integrity was unaffected by inhibition of cellular DNA replication and translation, however, disruption of transcription resulted in altered LRF localization. When compared to other cellular and viral foci LRF co-localized with the nuclear receptor co-activator GRIP1, while the HSV-1 gene products ICP4, ICP27 and VP13/14 disrupted foci formation to varying degrees. Interestingly, cells over-expressing LRF were resistant to productive HSV-1 infection and this resistance was dependent upon protein targeting and an N-terminal transactivation domain. When LRF knockdown cells were subjected to primary infection, HSV-1 gene expression and progeny virus yield were enhanced by ∼3 fold compared to wildtype cells. Taken together, these results indicate that LRF is a key regulator that may act direct or indirectly as a repressor of essential genes required for productive viral infection.

Abstract LB-333: A CREBZF/Zhangfei isoform activates CHOP and promotes apoptosis during prolonged endoplasmic reticulum stress

The basic leucine zipper transcription factor CREBZF (Zhangfei or ZF) was identified through its interaction with Herpes Simplex Virus-1 related cellular protein HCF-1, and has been implicated in cellular stress responses through its interaction with other proteins, such as CREB3/Luman and ATF4. Here we investigated the production of four CREBZF isoforms, which arise from translational initiation of a downstream AUG at codon 83 and mRNA alternative splicing that adds an IFFFR pentapeptidyl tail to the C-terminus. We found that in addition to transcriptional induction, the short-tailed CREBZF (stZF) isoform was specifically induced by prolonged ER stress treatment and amino acid deprivation. This stZF isoform is a potent transcriptional activator of the pro-apoptotic protein CHOP. Overexpression of stZF activates transcription of CHOP through a CCAAT enhancer binding protein (C/EBP)-ATF site, and promotes apoptosis. We propose that 1) CREBZF is a key component of the integrated stress response; 2) stZF is tightly regulated and induced primarily under prolonged cellular stress, and is essential for the role of CREBZF in inducing CHOP and promoting cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-333.