Rui Lu

Luman, a new member of the CREB/ATF family, binds to herpes simplex virus VP16-associated host cellular factor.

The human host cell factor (HCF) is expressed in a variety of adult and fetal tissues, and its gene is conserved in animals as diverse as mammals and insects. However, its only known function is to stabilize the herpes simplex virus virion transactivator VP16 in a complex with the cellular POU domain protein Oct-1 and cis-acting regulatory elements in promoters of immediate-early viral genes. To identify a cellular function for HCF, we used the yeast two-hybrid system to identify a cellular ligand for HCF. This protein, Luman, appears to be a cyclic AMP response element (CRE)-binding protein/activating transcription factor 1 protein of the basic leucine zipper superfamily. It binds CREs in vitro and activates CRE-containing promoters when transfected into COS7 cells. This activation of transcription was synergistically enhanced by the presence of CCAAT/enhancer-binding protein elements and inhibited by AP-1 elements in the promoter. In addition to a basic DNA binding domain, Luman possesses an unusually long leucine zipper and an acidic amino-terminal activation domain. These features in Luman are also present in what appear to be homologs in the mouse, Drosophila melanogaster, and Caenorhabditis elegans. Luman and VP16 appear to have similar mechanisms for binding HCF, as in vitro each competitively inhibited the binding of the other to HCF. In transfected cells, however, while VP16 strongly inhibited the ability of GAL-Luman to activate transcription from a GAL4 upstream activation sequence-containing promoter, Luman was unable to inhibit the activity of GAL-VP16. Luman appears to be a ubiquitous transcription factor, and its mRNA was detected in all human adult and fetal tissues examined. The possible role of HCF in regulating the function of this ubiquitous transcription factor is discussed.

Luman/CREB3 Induces Transcription of the Endoplasmic Reticulum (ER) Stress Response Protein Herp through an ER Stress Response Element

ABSTRACT Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER) membrane-bound transcription factor which is believed to undergo regulated intramembrane proteolysis in response to cellular cues. We previously found that Luman activates transcription from the unfolded protein response element. Here we report the identification of Herp, a gene involved in ER stress-associated protein degradation (ERAD), as a direct target of Luman. We found that Luman was transcriptionally induced and proteolytically activated by the ER stress inducer thaspsigargin. Overexpression of Luman activated transcription of cellular Herp via ER stress response element II (ERSE-II; ATTGG-N-CCACG) in the promoter region. Mutagenesis studies and chromatin immunoprecipitation assays showed that Luman physically associates with the Herp promoter, specifically the second half-site (CCACG) of ERSE-II. Luman was also necessary for the full activation of Herp during the ER stress response, since Luman small interfering RNA knockdown or functional repression by a dominant negative mutant attenuated Herp gene expression. Like Herp, overexpression of Luman protected cells against ER stress-induced apoptosis. With Luman structurally similar to ATF6 but resembling XBP1 in DNA-binding specificities, we propose that Luman is a novel factor that plays a role in ERAD and a converging point for various signaling pathways channeling through the ER.

Potential role for luman, the cellular homologue of herpes simplex virus VP16 (alpha gene trans-inducing factor), in herpesvirus latency.

ABSTRACT The cascade of herpes simplex virus (HSV) gene expression that results in viral replication begins with the activation of viral immediate-early (IE) genes by the virion-associated protein VP16. VP16 on its own is inefficient at associating with complexes formed on IE gene promoters and depends upon the cellular factor HCF for its activity. In this respect VP16 mimics the host basic leucine zipper (bZIP) protein Luman, which also requires HCF for activating transcription. Our objective is to explore interactions between Luman and HCF and to determine if they play a role in the biology of herpesviruses. In this report we show that in cultured cells ectopically expressed Luman was retained in the cytoplasm, where it colocalized with Calnexin, a protein normally associated with the endoplasmic reticulum (ER). Retention of Luman in the ER depends on a hydrophobic segment of the protein that probably serves as a transmembrane domain. Deletion of this domain changed the intracellular location of Luman so that most of the mutant protein was in the nucleus of cells. While HCF was present in the nucleus of most cells, in cells expressing Luman it was retained in the cytoplasm where the two proteins colocalized. This cytoplasmic association of Luman and HCF could also be demonstrated in neurons in trigeminal ganglia removed from cattle soon after death. Cells in tissue culture that expressed Luman, but not a mutant form of the protein that fails to bind HCF, were resistant to a productive infection with HSV type 1 (HSV-1). We hypothesize that similar Luman-HCF interactions in sensory neurons in trigeminal ganglia result in the suppression of viral replication and the establishment of latency. Interestingly, Luman could activate the promoters of IE110 and LAT, two genes that are critical for reactivation of HSV-1 from latency. This suggests a role for Luman in the reactivation process as well.

Cooperative interaction of Zhangfei and ATF4 in transactivation of the cyclic AMP response element

Zhangfei (ZF) is a basic region‐leucine zipper protein that has been implicated in herpesvirus infection cycle and related cellular processes. Here we show both in vivo and in vitro data demonstrating that ZF is a novel cellular binding partner of activating transcription factor 4 (ATF4) (or CREB2). We found that ZF competed with ATF4 to form ATF4‐ZF heterodimeric complexes through the bZIP regions. ZF enhanced ATF4 binding to the cAMP response element (CRE), and augmented activation of a CRE reporter by ATF4, in response to MEK1 activation. These results suggest an important role of ZF in the MEK1‐ATF4 signaling pathway.

A Novel Protein, Luman/CREB3 Reruitment Factor, Inhibits Luman Activation of the Unfolded Protein Response

ABSTRACT Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound cellular transcription factor. It has been implicated in the mammalian unfolded protein response (UPR), as well as herpes simplex virus reactivation from latency in sensory neurons. Here, we report the identification of a novel Luman recruitment factor (LRF). Like Luman, LRF is a UPR-responsive basic-region leucine zipper protein that is prone to proteasomal degradation. Being a highly unstable protein, LRF interacts with Luman through the leucine zipper region and promotes Luman degradation. LRF was found to recruit the nuclear form of Luman to discrete nuclear foci, which overlap with the nuclear receptor coactivator GRIP1 bodies, and repress the transactivation activity of Luman. Compared to LRF+/+ mouse embryonic fibroblast (MEF) cells, the levels of CHOP, EDEM, and Herp were elevated in LRF−/− MEF cells. We propose that LRF is a negative regulator of the UPR. For Luman, it may represent another level of regulation following Luman proteolytic cleavage on the ER and nuclear translocation. In addition to inducing rapid Luman turnover, LRF may repress the transactivation potential of Luman by sequestering it in the LRF nuclear bodies away from key cofactors (such as HCF-1) that are required for transcriptional activation.

Localization and functional characterization of pulmonary bovine odorant-binding protein.

Bovine odorant-binding protein (OBP) may function in olfaction and defense against oxidative injury, but its role in inflammation and defense against bacterial infection has not been investigated. Expression of OBP was discovered in the bovine lung and found to undergo changes in abundance during glucocorticoid administration and stress. OBP was localized to nasal, tracheal, and bronchial mucosal glands with immunohistochemistry, with faint expression in airway surface epithelium and none in bronchioles or alveoli. Two isoforms of OBP were identified, appearing to be differentially regulated during lipopolysaccharide-induced pulmonary inflammation, but differences between these isoforms were not revealed by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. Functional studies showed no effect of OBP on in vitro growth of Escherichia coli or Mannheimia haemolytica under iron-replete or iron-depleted conditions, nor did OBP opsonize bacteria for an enhanced neutrophil oxidative burst. However, OBP did reduce the ability of supernatants from lipopolysaccharide-stimulated macrophages to induce neutrophil chemotaxis. These findings indicate that OBP may inhibit neutrophil recruitment by inflammatory mediators, and they suggest an ability to bind macrophage-derived inflammatory mediators within the airways.