Balancing life and death: BCL-2 family members at diverse ER-mitochondrial contact sites.

The outer mitochondrial membrane is a busy place. One essential activity for cellular survival is the regulation of membrane integrity by the BCL-2 family of proteins. Another critical facet of the outer mitochondrial membrane is its close approximation with the endoplasmic reticulum. These mitochondrial-associated membranes (MAMs) occupy a significant fraction of the mitochondrial surface and serve as key signaling hubs for multiple cellular processes. Each of these pathways may be considered as forming their own specialized MAM subtype. Interestingly, like membrane permeabilization, most of these pathways play critical roles in regulating cellular survival and death. Recently, the pro-apoptotic BCL-2 family member BOK has been found within MAMs where it plays important roles in their structure and function. This has led to a greater appreciation that multiple BCL-2 family proteins, which are known to participate in numerous functions throughout the cell, also have roles within MAMs. In this review, we evaluate several MAM subsets, their role in cellular homeostasis, and the contribution of BCL-2 family members to their functions.

Yes, MAM!

Regulation of cell life and death by members of the BCL-2 family of proteins occurs at the mitochondria. Large portions of the mitochondria's outer membrane are found in tight approximation with the endoplasmic reticulum (ER), known as mitochondria-associated membranes (MAMs) or mitochondria-ER contact sites (MERCs). We recently reported that BOK is present within MAMs where it regulates Ca2+ transfer from the ER to the mitochondria, appropriate MAM components and MERC structure, and apoptosis.

BOK controls apoptosis by Ca2+ transfer through ER-mitochondrial contact sites.

Calcium transfer from the endoplasmic reticulum (ER) to mitochondria is a critical contributor to apoptosis. B cell lymphoma 2 (BCL-2) ovarian killer (BOK) localizes to the ER and binds the inositol 1,4,5-trisphosophate receptor (IP3R). Here, we show that BOK is necessary for baseline mitochondrial calcium levels and stimulus-induced calcium transfer from the ER to the mitochondria. Murine embryonic fibroblasts deficient for BOK have decreased proximity of the ER to the mitochondria and altered protein composition of mitochondria-associated membranes (MAMs), which form essential calcium microdomains. Rescue of the ER-mitochondrial juxtaposition with drug-inducible interorganelle linkers reveals a kinetic disruption, which when overcome in Bok-/- cells is still insufficient to rescue thapsigargin-induced calcium transfer and apoptosis. Likewise, a BOK mutant unable to interact with IP3R restores ER-mitochondrial proximity, but not ER-mitochondrial calcium transfer, MAM protein composition, or apoptosis. This work identifies the dynamic coordination of ER-mitochondrial contact by BOK as an important control point for apoptosis.

The Propagation and Quantification of Two Emerging Oncolytic Viruses: Vesicular Stomatitis (VSV) and Zika (ZIKV).

Developments in genetic engineering have allowed researchers and clinicians to begin harnessing viruses to target and kill cancer cells, either through direct lysis or through recruitment of antiviral immune responses. Two powerful viruses in the fight against cancer are the single-stranded RNA viruses vesicular stomatitis virus and Zika virus. Here, we describe methods to propagate and titer these two viruses. We also describe a simple cell-killing assay to begin testing modified viruses for increased potential killing of glioblastoma cells.

Minocycline mitigates the effect of neonatal hypoxic insult on human brain organoids.

Neonatal hypoxic injury (NHI) is a devastating cause of disease that affects >60% of babies born with a very low birth weight, resulting in significant morbidity and mortality, including life-long neurological consequences such as seizures, cerebral palsy, and intellectual disability. Hypoxic injury results in increased neuronal death, which disrupts normal brain development. Although animal model systems have been useful to study the effects of NHI, they do not fully represent the uniqueness and complexities of the human brain. To better understand the effects of hypoxia on human brain development, we have generated a brain organoid protocol and evaluated these cells over the course of 6 months. As anticipated, the expression of a forebrain marker, FOXG1, increased and then remained expressed over time, while there was a transition in the expression of the deep-layer (TBR1) and upper-layer (SATB2) cortical markers. In addition, ventral genes (Eng1 and Nkx2.1) as well as markers of specialized nonneuronal cells (Olig2 and GFAP) also increased at later time points. We next tested the development of our in vitro cerebral organoid model at different oxygen concentrations and found that hypoxia repressed gene markers for forebrain, oligodendrocytes, glial cells, and cortical layers, as well as genes important for the migration of cortical neurons. In contrast, ventral markers were either unaffected or even increased in expression with hypoxic insult. Interestingly, the negative effect of hypoxia on the dorsal brain genes as well as oligodendrocytes, and neuronal progenitors could be mitigated by the use of minocycline, an FDA-approved small molecule. Taken together, we have generated a unique and relevant in vitro human brain model system to study diseases such as NHI as well as their potential treatments. Using this system, we have shown the efficacy of minocycline for human NHI.

Inhibition of isoprenylation synergizes with MAPK blockade to prevent growth in treatment-resistant melanoma, colorectal, and lung cancer.

This study evaluates the use of HMG-CoA reductase inhibitors, or statins, as an adjunctive to BRAF and MEK inhibition as a treatment in melanomas and other tumors with driver mutations in the MAPK pathway. Experiments used simvastatin in conjunction with vemurafenib and selumetinib in vitro and simvastatin with vemurafenib in vivo to demonstrate additional growth abrogation beyond MAPK blockade alone. Additional studies demonstrated that statin anti-tumor effects appeared to depend on inhibition of isoprenoid synthesis given rescue with add-back of downstream metabolites. Ultimately, we concluded that statins represent a possible useful adjunctive therapy in MAPK-driven tumors when given with current approved targeted therapy.

Combinatorial Screening of Pancreatic Adenocarcinoma Reveals Sensitivity to Drug Combinations Including Bromodomain Inhibitor Plus Neddylation Inhibitor.

Pancreatic adenocarcinoma (PDAC) is the fourth most common cause of cancer-related death in the United States. PDAC is difficult to manage effectively, with a five-year survival rate of only 5%. PDAC is largely driven by activating KRAS mutations, and as such, cannot be directly targeted with therapeutic agents that affect the activated protein. Instead, inhibition of downstream signaling and other targets will be necessary to effectively manage PDAC. Here, we describe a tiered single-agent and combination compound screen to identify targeted agents that impair growth of a panel of PDAC cell lines. Several of the combinations identified from the screen were further validated for efficacy and mechanism. Combination of the bromodomain inhibitor JQ1 and the neddylation inhibitor MLN4294 altered the production of reactive oxygen species in PDAC cells, ultimately leading to defects in the DNA damage response. Dual bromodomain/neddylation blockade inhibited in vivo growth of PDAC cell line xenografts. Overall, this work revealed novel combinatorial regimens, including JQ1 plus MLN4294, which show promise for the treatment of RAS-driven PDAC. Mol Cancer Ther; 16(6); 1041-53. ©2017 AACR.

Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake.

Cholesterol is a lipid that is critical for steroid hormone production and the integrity of cellular membranes, and, as such, it is essential for cell growth. The epidermal growth factor receptor (EGFR) family member ERBB4, which forms signaling complexes with other EGFR family members, can undergo ligand-induced proteolytic cleavage to release a soluble intracellular domain (ICD) that enters the nucleus to modify transcription. We found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance low-density lipoprotein (LDL) uptake and cholesterol biosynthesis. Expression of the ERBB4 ICD in mammary epithelial cells or activation of ERBB4 with the ligand neuregulin 1 (NRG1) induced the expression of SREBP target genes involved in cholesterol biosynthesis, including HMGCR and HMGCS1, and lipid uptake, LDLR, which encodes the LDL receptor. Addition of NRG1 increased the abundance of the cleaved, mature form of SREBP-2 through a pathway that was blocked by addition of inhibitors of PI3K (phosphatidylinositol 3-kinase) or dual inhibition of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, but not by inhibition of AKT or mTORC1. Pharmacological inhibition of the activity of SREBP site 1 protease or of all EGFR family members (with lapatinib), but not EGFR alone (with erlotinib), impaired NRG1-induced expression of cholesterol biosynthesis genes. Collectively, our findings indicated that activation of ERBB4 promotes SREBP-2-regulated cholesterol metabolism. The connections of EGFR and ERBB4 signaling with SREBP-2-regulated cholesterol metabolism are likely to be important in ERBB-regulated developmental processes and may contribute to metabolic remodeling in ERBB-driven cancers.

The Membrane Associated RING-CH Proteins: A Family of E3 Ligases with Diverse Roles through the Cell.

Since the discovery that conjugation of ubiquitin to proteins can drive proteolytic degradation, ubiquitination has been shown to perform a diverse range of functions in the cell. It plays an important role in endocytosis, signal transduction, trafficking of vesicles inside the cell, and even DNA repair. The process of ubiquitination-mediated control has turned out to be remarkably complex, involving a diverse array of proteins and many levels of control. This review focuses on a family of structurally related E3 ligases termed the membrane-associated RING-CH (MARCH) ubiquitin ligases, which were originally discovered as structural homologs to the virals E3s, K3, and K5 from Kaposi's sarcoma-associated herpesvirus (KSHV). These proteins contain a catalytic RING-CH finger and are typically membrane-bound, with some having up to 14 putative transmembrane domains. Despite several lines of evidence showing that the MARCH proteins play a complex and essential role in several cellular processes, this family remains understudied.

Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins down regulate both DC-SIGN and DC-SIGNR.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of multicentric Castleman's disease, primary effusion lymphoma and Kaposi's sarcoma. In this study, we show that like the C-type lectin DC-SIGN, the closely related DC-SIGNR can also enhance KSHV infection. Following infection, they are both targeted for down modulation and our data indicate that the KSHV MARCH-family ubiquitin ligase K5 is mediating this regulation and subsequent targeting for degradation of DC-SIGN and DC-SIGNR in the context of the virus. The closely related viral K3 protein, is also able to target these lectins in exogenous expressions studies, but only weakly during viral infection. In addition to requiring a functional RING-CH domain, several protein trafficking motifs in the C-terminal region of both K3 and K5 are important in regulation of DC-SIGN and DC-SIGNR. Further exploration of this modulation revealed that DC-SIGN is endocytosed from the cell surface in THP-1 monocytes, but degraded from an internal location with minimal endocytosis in HEK-293 cells. Pull-down data indicate that both K3 and K5 preferentially associate with immature forms of the lectins, mediating their ubiquitylation and degradation. Together, these data emphasize the molecular complexities of K3 and K5, while expanding the repertoire of targets of these two viral proteins.

The MARCH family E3 ubiquitin ligase K5 alters monocyte metabolism and proliferation through receptor tyrosine kinase modulation.

Kaposi's sarcoma (KS) lesions are complex mixtures of KS-associated herpesvirus (KSHV)-infected spindle and inflammatory cells. In order to survive the host immune responses, KSHV encodes a number of immunomodulatory proteins, including the E3 ubiquitin ligase K5. In exploring the role of this viral protein in monocytes, we made the surprising discovery that in addition to a potential role in down regulation of immune responses, K5 also contributes to increased proliferation and alters cellular metabolism. This ubiquitin ligase increases aerobic glycolysis and lactate production through modulation of cellular growth factor-binding receptor tyrosine kinase endocytosis, increasing the sensitivity of cells to autocrine and paracrine factors. This leads to an altered pattern of cellular phosphorylation, increases in Akt activation and a longer duration of Erk1/2 phosphorylation. Overall, we believe this to be the first report of a virally-encoded ubiquitin ligase potentially contributing to oncogenesis through alterations in growth factor signaling cascades and opens a new avenue of research in K5 biology.

Placental viral infection sensitizes to endotoxin-induced pre-term labor: a double hit hypothesis.

PROBLEM: Among pregnant women, acquired viral infections with a concurrent bacterial infection is a detrimental factor associated to poor prognosis. We evaluate the effect of a viral infection that does not lead to pre-term labor on the response to low doses of lipopolysaccharide (LPS). Our objectives were (i) to characterize the effect of a viral infection concurrent with exposure to microbial products on pregnancy outcome and (ii) to characterize the placental and fetal immune responses to the viral sensitization to LPS. METHOD: C57B/6 wild-type mice were injected with murine gammaherpesvirus 68 (MHV68) at E8.5. Either PBS or LPS was injected i.p. at E15.5. Pregnancy outcome and cytokine/chemokine profile from implantation sites were analyzed by multiplex. RESULTS: LPS treatment of MHV-68-infected animals induced pre-term delivery and fetal death in 100% of the mice. Pre-term labor was characterized by a upregulation of pro-inflammatory cytokines and chemokines in both placenta and decidua. Similar profiles were observed from MHV-68-infected human primary trophoblast and trophoblast cell lines in response to LPS. CONCLUSION: We describe for the first time that a sub-clinical viral infection in pregnant mice might sensitize to a bacterial infection leading to pre-term delivery. We propose the 'Double Hit Hypothesis' where the presence of a viral infection enhances the effect of bacterial products during pregnancy leading not only to pre-term labor but likely larger adverse outcomes.

Characterization of cytoplasmic motifs important in rhesus rhadinovirus gB processing and trafficking.

Rhesus monkey rhadinovirus (RRV) is highly related to Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus etiologically-linked with several cancers. Glycoprotein B (gB) homologues are encoded by all herpesviruses and play a role in virus attachment, entry, and in egress. We have found that RRV gB, like KSHV gB, is cleaved at a consensus furin cleavage site and is modified by both N-linked and O-linked glycosylation. Mutagenesis of three tyrosine- based trafficking motifs, a diacidic tyrosine motif, and a di-lucine motif in the cytoplasmic region revealed a role for these sequences in both ER export and endocytosis from the plasma membrane. These experiments provide a basis for further experiments looking at gB incorporation and role in gamma-herpesvirus assembly and egress.

Characterization of the rhesus fibromatosis herpesvirus MARCH family member rfK3.

Retroperitoneal fibromatosis-associated herpesvirus (RFHV) is a gamma-herpesvirus of macaques that is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV). Herein, we present characterization of the K3 gene from RFHV, a homologue of the KSHV K3 and K5 genes. Like the KSHV proteins, kK3 and kK5, the rfK3 protein decreases cell surface MHC I levels. Similar to kK5, rfK3 also modulates ICAM-1, but not another kK5 target, B7.2. Inhibitors of dynamin or mutations in the rfK3 RING-CH E3 ubiquitin ligase domain block cellular target regulation, implicating a ubiquitin-dependent, endocytosis-mediated mechanism for target down regulation and degradation. Overall, this manuscript presents the first characterization of a non-human primate virus MARCH family E3 ubiquitin ligase contributing important information about the evolution of immune avoidance strategies in primate viruses and paving the way for an animal model examining the importance of kK3 and kK5 in vivo.

The Kaposi's sarcoma-associated herpesvirus K5 E3 ubiquitin ligase modulates targets by multiple molecular mechanisms.

Kaposi's sarcoma-associated herpesvirus encodes two highly related membrane-associated, RING-CH-containing (MARCH) family E3 ubiquitin ligases, K3 and K5, that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report we present data that while K5 modulation of major histocompatibility complex class I (MHC-I) closely mirrors the mechanisms used by K3, alternative molecular pathways are utilized by this E3 ligase in the down regulation of intercellular adhesion molecule 1 (ICAM-1) and B7.2. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct an increased rate of MHC-I or ICAM-1 internalization but still able to down regulate B7.2 in a ubiquitin-dependent but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC-I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. These data indicate that while K5 can act as an E3 ubiquitin ligase to directly mediate cell surface molecule destruction, regulation of its targets occurs through multiple pathways, including ubiquitin-independent mechanisms.

Kaposi sarcoma herpesvirus K5 removes CD31/PECAM from endothelial cells.

The transmembrane ubiquitin ligase K5/MIR2 of Kaposi sarcoma herpesvirus (KSHV) mediates internalization and lysosomal degradation of glycoproteins involved in antigen presentation and co-stimulation. In endothelial cells (ECs), K5 additionally reduced expression of CD31/platelet-endothelial cell adhesion molecule (PECAM), an adhesion molecule regulating cell-cell interactions of ECs, platelets, monocytes, and T cells. K5 also reduced EC migration, a CD31-dependent process. Unlike other K5 substrates, both newly synthesized and pre-existing CD31 molecules were targeted by K5. K5 was transported to the cell surface and ubiquitinated pre-existing CD31, resulting in endocytosis and lysosomal degradation. In the endoplasmic reticulum, newly synthesized CD31 was degraded by proteasomes, which required binding of phosphofurin acidic cluster sorting protein-2 (PACS-2) to acidic residues in the carboxyterminal tail of K5. Thus, CD31, a novel target of K5, is efficiently removed from ECs by a dual degradation mechanism that is regulated by the subcellular sorting of the ubiquitin ligase. K5-mediated degradation of CD31 is likely to affect EC function in KS tumors.

Activation of CD21 and CD23 gene expression by Kaposi's sarcoma-associated herpesvirus RTA.

Epstein-Barr virus (EBV) EBNA2 and Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) are recruited to their responsive elements through interaction with a Notch-mediated transcription factor, RBP-Jkappa. In particular, RTA and EBNA2 interactions with RBP-Jkappa are essential for the lytic replication of KSHV and expression of B-cell activation markers CD21 and CD23a, respectively. Here, we demonstrate that like EBV EBNA2, KSHV RTA strongly induces CD21 and CD23a expression through RBP-Jkappa binding sites in the first intron of CD21 and in the CD23a core promoter, respectively. However, unlike EBV EBNA2, which alters immunoglobulin mu (Igmu) and c-myc gene expression, RTA did not affect Igmu and c-myc expression, indicating that KSHV RTA targets the Notch signal transduction pathway in a manner similar to but distinct from that of EBV EBNA2. Furthermore, RTA-induced expression of CD21 glycoprotein, which is an EBV receptor, efficiently facilitated EBV infection. In addition, RTA-induced CD23 glycoprotein underwent proteolysis and gave rise to soluble CD23 (sCD23) molecules in B lymphocytes and KSHV-infected primary effusion lymphocytes. sCD23 then stimulated primary human lymphocytes. These results demonstrate that cellular CD21 and CD23a are common targets for B lymphotropic gammaherpesviruses and that KSHV RTA regulates RBP-Jkappa-mediated cellular gene expression, which ultimately provides a favorable milieu for viral reproduction in the infected host.

Characterization of the Herpesvirus saimiri Orf51 protein.

Herpesvirus saimiri (HVS) is a gamma(2)-herpesvirus sharing genomic colinearity and a high degree of functional homology with HHV-8. To begin exploring the correlates of HVS infectivity and neutralization, we designed and implemented a new reporter assay. Using this assay, we could demonstrate that HVS neutralizing antibodies are present at high levels in naturally infected squirrel monkeys and are strongly induced after pathogenic, experimental infection of common marmosets. Further, we demonstrated that viral entry is influenced by cellular glycosaminoglycans and that, similar to HHV-8, soluble heparin is capable of blocking infectivity. We next cloned and characterized the positional homologue of HHV-8 K8.1, HVS Orf51. N-glycosidase F treatment indicates that like K8.1, Orf51 is a glycoprotein. Found in the viral particle, it localizes to the endoplasmic reticulum of expressing cells. Like K8.1, Orf51 could bind to agarose-conjugated heparin, implicating this molecule in viral attachment to cells. These studies provide the groundwork for additional experiments into the role that this protein may be playing in viral pathogenicity, persistence, and cell tropism.

Herpesviral protein targets a cellular WD repeat endosomal protein to downregulate T lymphocyte receptor expression.

Herpesvirus saimiri Tip associates with Lck and downregulates Lck signal transduction. Here we demonstrate that Tip targets a lysosomal protein p80, which consists of an N-terminal WD repeat domain and a C-terminal coiled-coil domain. Interaction of Tip with p80 facilitated lysosomal vesicle formation and subsequent recruitment of Lck into the lysosomes for degradation. Consequently, Tip interactions with Lck and p80 result in downregulation of T cell receptor (TCR) and CD4 surface expression. Remarkably, these actions of Tip are functionally and genetically separable: the N-terminal p80 interaction is responsible for TCR downregulation and the C-terminal Lck interaction is responsible for CD4 downregulation. Thus, lymphotropic herpesvirus has evolved an elaborate mechanism to deregulate lymphocyte receptor expression to disarm host immune control.

Kaposi's sarcoma associated herpesvirus immune evasion strategies.

The battle between viruses and their hosts is beautiful in its complexity. The interplay between viral proteins and the immune system has taught researchers much about not just the virus, but also the molecular mechanisms underlying the immune response. With additional evasion strategies constantly being described, this avenue of research is still rich with potential discoveries. In this review, we examine a number of proteins encoded by Kaposi's sarcoma herpesvirus (HHV-8) and detail how they aid the virus in escape from immune system elimination. We include, where possible, examples from other homologous viral systems.