Tyson V. Sharp

K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function

ABSTRACT The Kaposi’s sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, ∼50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman’s disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.

Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin

We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposis sarcoma‐associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin‐ΔEx3, a splice variant of human survivin that protects cells from apoptosis by an undefined mechanism. Both K7 and survivin‐ΔEx3 contain a mitochondrial‐targeting sequence, an N‐terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl‐2 homology)‐like domain. These suggested that K7 is a new viral anti‐apoptotic protein and survivin‐ΔEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl‐2 resides. K7 does not associate with Bax, but does bind to Bcl‐2 via its putative BH2 domain. In addition, K7 binds to active caspase‐3 via its BIR domain and thus inhibits the activity of caspase‐3. The BH2 domain of K7 is crucial for the inhibition of caspase‐3 activity and is therefore essential for its anti‐apoptotic function. Furthermore, K7 bridges Bcl‐2 and activated caspase‐3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti‐apoptotic complex that presents effector caspases to Bcl‐2, enabling Bcl‐2 to inhibit caspase activity. These data also suggest that survivin‐ΔEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitor‐of‐apoptosis protein).

The M Type K15 Protein of Kaposi's Sarcoma-Associated Herpesvirus Regulates MicroRNA Expression via Its SH2-Binding Motif To Induce Cell Migration and Invasion

ABSTRACT Kaposis sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS. In vivo, KS is a tumor capable of spreading throughout the body, and pulmonary metastasis is observed clinically. In vitro, KSHV induces the invasiveness of endothelial cells. The KSHV open reading frame K15 is a KSHV-specific gene encoding a transmembrane protein. Two highly divergent forms of K15, the predominant (P) and minor (M) forms (K15P and K15M, respectively), have been identified in different KSHV strains. The two K15 alleles resemble the latent membrane protein 2A (LMP2A) gene of Epstein-Barr virus (EBV) in their genomic locations and protein topology. Also, both K15 proteins have motifs similar to those found in the EBV LMP1 protein. K15 therefore appears to be a hybrid of a distant evolutionary relative of EBV LMP1 and LMP2A. Since both LMP1 and LMP2A proteins are capable of inducing cell motility, we sought to determine whether K15 has similar abilities. In this study, we show that K15M is latently expressed in KSHV-positive PEL cells and knockdown of K15M in PEL cells reduces cell motility. K15M localizes to lysosomal membranes and induces cell migration, invasion, and NF-κB (but not AP-1) activity via its conserved SH2-binding motif. K15M also induces the expression of microRNAs miR-21 and miR-31 via this conserved motif, and knocking down both these microRNAs eliminates K15M-induced cell motility. Therefore, K15M may contribute to KSHV-mediated tumor metastasis and angiogenesis via regulation of miR-21 and miR-31, which we show here for the first time to be a specific regulator of cell migration. In light of these findings, the targeting of K15 or the downstream microRNAs regulated by it may represent novel therapies for treatment of KSHV-associated neoplasia.

Regulation of the interferon-inducible eIF-2α protein kinase by small RNAs

This review describes the structure and function of the double-stranded RNA-dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered.

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.

SMG-1 and mTORC1 act antagonistically to regulate response to injury and growth in planarians

Planarian flatworms are able to both regenerate their whole bodies and continuously adapt their size to nutrient status. Tight control of stem cell proliferation and differentiation during these processes is the key feature of planarian biology. Here we show that the planarian homolog of the phosphoinositide 3-kinase-related kinase (PIKK) family member SMG-1 and mTOR complex 1 components are required for this tight control. Loss of smg-1 results in a hyper-responsiveness to injury and growth and the formation of regenerative blastemas that remain undifferentiated and that lead to lethal ectopic outgrowths. Invasive stem cell hyper-proliferation, hyperplasia, hypertrophy, and differentiation defects are hallmarks of this uncontrolled growth. These data imply a previously unappreciated and novel physiological function for this PIKK family member. In contrast we found that planarian members of the mTOR complex 1, tor and raptor, are required for the initial response to injury and blastema formation. Double smg-1 RNAi experiments with tor or raptor show that abnormal growth requires mTOR signalling. We also found that the macrolide rapamycin, a natural compound inhibitor of mTORC1, is able to increase the survival rate of smg-1 RNAi animals by decreasing cell proliferation. Our findings support a model where Smg-1 acts as a novel regulator of both the response to injury and growth control mechanisms. Our data suggest the possibility that this may be by suppressing mTOR signalling. Characterisation of both the planarian mTORC1 signalling components and another PIKK family member as key regulators of regeneration and growth will influence future work on regeneration, growth control, and the development of anti-cancer therapies that target mTOR signalling.

The chromosome 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer development

Loss of heterozygosity (LOH) and homozygous deletions at chromosome 3p21.3 are common in both small and nonsmall cell lung cancers, indicating the likely presence of tumor suppressor genes (TSGs). Although genetic and epigenetic changes within this region have been identified, the functional significance of these changes has not been explored. Concurrent protein expression and genetic analyses of human lung tumors coupled with functional studies have not been done. Here, we show that expression of the 3p21.3 gene, LIMD1, is frequently down-regulated in human lung tumors. Loss of LIMD1 expression occurs through a combination of gene deletion, LOH, and epigenetic silencing of transcription without evidence for coding region mutations. Experimentally, LIMD1 is a bona fide TSG. Limd1−/− mice are predisposed to chemical-induced lung adenocarcinoma and genetic inactivation of Limd1 in mice heterozygous for oncogenic K-RasG12D markedly increased tumor initiation, promotion, and mortality. Thus, we conclude that LIMD1 is a validated chromosome 3p21.3 tumor-suppressor gene involved in human lung cancer development. LIMD1 is a LIM domain containing adapter protein that localizes to E-cadherin cell–cell adhesive junctions, yet also translocates to the nucleus where it has been shown to function as an RB corepressor. As such, LIMD1 has the potential to communicate cell extrinsic or environmental cues with nuclear responses.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex.

The role of hypoxia in stem cell potency and differentiation

Regenerative medicine relies on harnessing the capacity of stem cells to grow, divide and differentiate safely and predictably. This may be in the context of expanding stem cells in vitro or encouraging their expansion, mobilization and capacity to regenerate tissues either locally or remotely in vivo. In either case, understanding the stem cell niche is fundamental to recapitulating or manipulating conditions to enable therapy. It has become obvious that hypoxia plays a fundamental role in the maintenance of the stem cell niche. Low O2 benefits the self-renewal of human embryonic, hematopoietic, mesenchymal and neural stem cells, as well as improving the efficiency of genetic reprogramming to induced pluripotency. There is emerging evidence that harnessing or manipulating the hypoxic response can result in safer, more efficacious methodologies for regenerative medicine.

Functional Expression of Secreted Proteins from a Bicistronic Retroviral Cassette Based on Foot-and-Mouth Disease Virus 2A Can Be Position Dependent

The expression of two or more genes from a single viral vector has been widely used to label or select for cells containing the transgenic element. Identification of the foot-and-mouth disease virus (FMDV) 2A cleavage peptide as a polycistronic linker capable of producing equivalent levels of transgene expression has greatly improved this approach in the field of gene therapy. However, as a consequence of 2A posttranslational cleavage the upstream protein is left with a residual 19 amino acids from the 2A sequence on its carboxy terminus, and the downstream protein is left with an additional 2 to 5 amino acids on its amino terminus. Here we have assessed the functional consequences of the FMDV 2A cleavage motif on two secreted proteins (interleukin [IL]-2 and transforming growth factor [TGF]-β) when expressed from a retroviral bicistronic vector. Whereas IL-2 expression and function were found to be unaffected by the 2A motif in either orientation, functional expression of secreted TGF-β was significantly abrogated when the transgene was expressed upstream of the 2A sequence. We believe this is a consequence of aberrant cleavage and intracellular trafficking of the TGF-β polyprotein. These results highlight that to achieve functional expression of secreted proteins consideration must be taken of the transgenic proteins posttranslational modification and trafficking when using 2A-based bicistronic cassettes.