Discovery of small-molecule positive allosteric modulators of Parkin E3 ligase.

Pharmacological activation of the E3 ligase Parkin represents a rational therapeutic intervention for the treatment of Parkinson's disease. Here we identify several compounds that enhance the activity of wildtype Parkin in the presence of phospho-ubiquitin and act as positive allosteric modulators (PAMs). While these compounds activate Parkin in a series of biochemical assays, they do not act by thermally destabilizing Parkin and fail to enhance the Parkin translocation rate to mitochondria or to enact mitophagy in cell-based assays. We conclude that in the context of the cellular milieu the therapeutic window to pharmacologically activate Parkin is very narrow.

Association of Kaposi's Sarcoma-Associated Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene Expression.

Transcription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes.

Is the Modified Early Warning Score able to enhance clinical observation to detect deteriorating patients earlier in an Accident & Emergency Department?

BACKGROUND: Currently there is no study to prove the accuracy of any early warning system on a group of patients who are waiting for in-patient beds in emergency department (ED). The study objective is to compare the performances of detecting patient deterioration with and without using the Modified Early Warning Score for a group of patients who are waiting for in-patient beds in a public ED. METHODS: An observational study was conducted in a public ED in Hong Kong between January and March 2013. During this period, the Modified Early Warning Score (MEWS) was incorporated into current nursing practice for patient monitoring in selected days. Nurses then made decision according to the MEWS to notify senior nurses or senior doctors of patient deterioration. There was a comparison group with target patients being monitored without using the MEWS. RESULTS: A total of 545 patients were recruited, with 269 patients in the MEWS group. Using the MEWS for patient observation had a 100% sensitivity and a 98.3% specificity in detecting patient deterioration, while there was also a high sensitivity and a high specificity (100% and 97.8%) in the comparison group. The findings also showed that respiratory rate was a significant vital sign to detect patient deterioration. CONCLUSIONS: Using the MEWS for patient monitoring did not significantly enhance the performance in detecting patient deterioration for a group of patients who are waiting for in-patient beds in a public ED. However, the MEWS may be beneficial to less experienced nurses who have less clinical experience to identify patient deterioration.

Negative regulation of NF-κB activity by brain-specific TRIpartite Motif protein 9.

The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The ß-transducin repeat-containing protein (ß-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. ß-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation. Here we report that TRIM9 is a negative regulator of NF-κB activation. Interaction between the phosphorylated degron motif of TRIM9 and the WD40 repeat region of ß-TrCP prevented ß-TrCP from binding its substrates, stabilizing IκBα and p100 and thereby blocking NF-κB activation. Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production. This study not only elucidates a mechanism for TRIM9-mediated regulation of the ß-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

Kaposi's sarcoma-associated herpesvirus K3 and K5 ubiquitin E3 ligases have stage-specific immune evasion roles during lytic replication.

UNLABELLED: The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a ΔK3 ΔK5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1α-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFP(high) and pK8.1-driven blue fluorescent protein-positive (BFP(+)) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages. IMPORTANCE: Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.

Pulse Chase of Suspension Cells.

Pulse-chase method is a powerful technique used to follow the dynamics of proteins over a period of time. The expression level, processing, transport, secretion or half-life of proteins can be tracked by metabolically labeling the cells, such as with radiolabeled amino acids (pulse step). This protocol describes the condition used to study the folding and disulfide bond formation of immunoglobulin in suspension cells. With some minor modifications, this protocol can be adapted to study the degradation rate or the secretion of target proteins.

The product of Kaposi's sarcoma-associated herpesvirus immediate early gene K4.2 regulates immunoglobulin secretion and calcium homeostasis by interacting with and inhibiting pERP1.

Chaperones are proteins that assist the noncovalent folding and assembly of macromolecular polypeptide chains, ultimately preventing the formation of nonfunctional or potentially toxic protein aggregates. Plasma cell-induced-endoplasmic reticulum (ER)-resident protein 1 (pERP1) is a cellular chaperone that is preferentially expressed in marginal-zone B cells and is highly upregulated during plasma cell differentiation. While initially identified as a dedicated factor for the assembly of secreted IgM, pERP1 has since been implicated in suppressing calcium mobilization, and its expression is misregulated in multiple tumors. A number of herpesvirus immediate early gene products play important roles in the regulation of viral gene expression and/or evasion of host immune responses. Here, we report that the Kaposi's sarcoma-associated herpesvirus (KSHV) immediate early viral gene K4.2 encodes an endoplasmic reticulum-localized protein that interacts with and inhibits pERP1. Consequently, K4.2 expression interfered with immunoglobulin secretion by delaying the kinetics of immunoglobulin assembly and also led to increased responsiveness of B-cell receptor signal transduction by enhancing phosphotyrosine signals and intracellular calcium fluxes. Furthermore, K4.2 expression also appeared to contribute to maximal lytic replication by enhancing viral glycoprotein expression levels and ultimately promoting infectious-virus production. Finally, immunohistochemistry analysis showed that pERP1 expression was readily detected in KSHV-positive cells from multicentric Castleman's disease (MCD) and Kaposi's sarcoma (KS) lesions, suggesting that pERP1 may have potential roles in the KSHV life cycle and malignancy. In conclusion, our data suggest that K4.2 participates in lytic replication by enhancing calcium flux and viral glycoprotein expression, but also by interfering with immunoglobulin assembly to potentially dampen the adaptive immune response.

Novel Phosphorylations of IKKγ/NEMO.

Central to NF-κB signaling pathways is IKKγ/NEMO, a regulatory subunit of the cytoplasmic IκB kinase (IKK) complex, which undergoes various posttranslational modifications, specifically phosphorylation, to regulate its function. Furthermore, Kaposi's sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1ß (IL-1ß) converting enzyme (FLICE) inhibitory protein (vFLIP) activates the NF-κB signaling pathway by directly interacting with IKKγ/NEMO. However, the exact functions of IKKγ/NEMO phosphorylation and its KvFLIP interaction in NF-κB activation remain elusive. Here, we report two novel phosphorylation sites of IKKγ/NEMO and their negative effect on the IKKγ/NEMO-mediated NF-κB signaling pathway. First, the Src family protein tyrosine kinases (SF-PTKs), including Src, Fyn, Lyn, and Fgr, interact with and phosphorylate tyrosine residue 374 (Y374) of IKKγ/NEMO. Mutation of the Y374 residue to phenylalanine (Y374F) specifically abolished SF-PTK-mediated tyrosine phosphorylation, leading to increased tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Moreover, our mass spectrometry analysis found that the serine 377 residue (S377) of IKKγ/NEMO underwent robust phosphorylation upon KvFLIP expression. Replacement of the IKKγ/NEMO S377 residue by alanine (S377A) or glutamic acid (S377E) resulted in a significant increase or decrease of NF-κB activity and TNF-α-mediated IL-6 cytokine production, respectively. Our study thus demonstrates that the Y374 or S377 residue located at the C-terminal proline-rich domain of human IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This study suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway. IMPORTANCE Since unchecked regulation of NF-κB has been linked to uncontrolled proliferation and cell death, the downregulation of the NF-κB signaling pathway is as important as its activation. Specifically, the phosphorylation-mediated modification of IKKγ/NEMO is a critical regulatory mechanism of NF-κB activity. Here, we report two novel phosphorylations of IKKγ/NEMO and their negative effects on the NF-κB signaling pathway. First, the Src family protein tyrosine kinase interacts with and phosphorylates tyrosine residue 374 of IKKγ/NEMO, suppressing tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Additionally, Kaposi's sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1ß (IL-1ß) converting enzyme (FLICE) inhibitory protein (KvFLIP) expression induces a robust phosphorylation of the serine 377 residue of IKKγ/NEMO, resulting in a significant decrease of NF-κB activity. Our study thus demonstrates that the Y374 or S377 residue of IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This also suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway.

Construction and manipulation of a new Kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome clone.

Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (∼5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.

Negative elongation factor-mediated suppression of RNA polymerase II elongation of Kaposi's sarcoma-associated herpesvirus lytic gene expression.

Genome-wide chromatin immunoprecipitation assays indicate that the promoter-proximal pausing of RNA polymerase II (RNAPII) is an important postinitiation step for gene regulation. During latent infection, the majority of Kaposi's sarcoma-associated herpesvirus (KSHV) genes is silenced via repressive histone marks on their promoters. Despite the absence of their expression during latency, however, several lytic promoters are enriched with activating histone marks, suggesting that mechanisms other than heterochromatin-mediated suppression contribute to preventing lytic gene expression. Here, we show that the RNAPII-mediated transcription of the KSHV OriLytL, K5, K6, and K7 (OriLytL-K7) lytic genes is paused at the elongation step during latency. Specifically, the RNAPII-mediated transcription is stalled by the host's negative elongation factor (NELF) at the promoter regions of OriLytL-K7 lytic genes during latency, leading to the hyperphosphorylation of the serine 5 residue and the hypophosphorylation of the serine 2 of the C-terminal domain of the RNAPII large subunit, a hallmark of stalled RNAPII. Consequently, depletion of NELF expression induced transition of stalled RNAPII into a productive transcription elongation at the promoter-proximal regions of OriLytL-K7 lytic genes, leading to their RTA-independent expression. Using an RTA-deficient recombinant KSHV, we also showed that expression of the K5, K6, and K7 lytic genes was highly inducible upon external stimuli compared to other lytic genes that lack RNAPII on their promoters during latency. These results indicate that the transcription elongation of KSHV OriLytL-K7 lytic genes is inhibited by NELF during latency, but can also be promptly reactivated in an RTA-independent manner upon external stimuli.

Inhibition of RIG-I-mediated signaling by Kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase ORF64.

Virus infection triggers interferon (IFN)-mediated innate immune defenses in part through viral nucleic acid interactions. However, the immune recognition mechanisms by which the host identifies incoming DNA viruses are still elusive. Here, we show that increased levels of Kaposi's sarcoma-associated herpesvirus (KSHV) persistency are observed in retinoic acid-inducible gene I (RIG-I)-deficient cells and that KSHV ORF64, a tegument protein with deubiqutinase (DUB) activity, suppresses RIG-I-mediated IFN signaling by reducing the ubiquitination of RIG-I, crucial for its activation. This study suggests that RIG-I plays a potential role in sensing KSHV infection and that KSHV ORF64 DUB counteracts RIG-I signaling.

Linear ubiquitin assembly complex negatively regulates RIG-I- and TRIM25-mediated type I interferon induction.

Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway.

Epigenetic analysis of KSHV latent and lytic genomes.

Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle.

Chromatin remodeling and repair of DNA double-strand breaks.

Eukaryotic cells have developed conserved mechanisms to efficiently sense and repair DNA damage that results from constant chromosomal lesions. DNA repair has to proceed in the context of chromatin, and both histone-modifiers and ATP-dependent chromatin remodelers have been implicated in this process. Here, we review the current understanding and new hypotheses on how different chromatin-modifying activities function in DNA repair in yeast and metazoan cells.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen induces a strong bend on binding to terminal repeat DNA.

During latency, the Kaposi's sarcoma-associated herpesvirus genome is maintained as a circular episome, replicating in synchrony with host chromosomes. Replication requires the latency-associated nuclear antigen (LANA) and an origin of latent DNA replication located in the viral terminal repeats, consisting of two LANA binding sites (LBSs) and a GC-rich sequence. Here, we show that the recruitment of a LANA dimer to high-affinity site LBS-1 bends DNA by 57 degrees and towards the major groove. The cooccupancy of LBS-1 and lower-affinity LBS-2 induces a symmetrical bend of 110 degrees . By changing the origin architecture, LANA may help to assemble a specific nucleoprotein structure important for the initiation of DNA replication.

Transcriptional activation by the Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen is facilitated by an N-terminal chromatin-binding motif.

In immunocompromised patients, infection with Kaposi's sarcoma-associated herpesvirus (KSHV) can give rise to Kaposi's sarcoma and several lymphoproliferative disorders. In these tumors, KSHV establishes a latent infection in many of the rapidly proliferating and morphologically abnormal cells. Only a few viral gene products are expressed by the latent virus, and one of the best characterized is the latency-associated nuclear antigen (LANA), a nuclear protein required for the maintenance of viral episomal DNA in the dividing host cell. LANA can also activate or repress an assortment of cellular and viral promoters and may contribute to pathogenesis by allowing the proliferation and survival of host cells. Here we show that activation of the human E2F1 and cyclin-dependent kinase-2 (CDK2) promoters requires elements from both the N- and C-terminal regions of LANA. Deletion of the first 22 amino acids, which are necessary for episome tethering, does not affect nuclear localization but significantly reduces transactivation. Within the deleted peptide, we have identified a short sequence, termed the chromatin-binding motif (CBM), that binds tightly to interphase and mitotic chromatin. A second chromatin-binding activity resides in the C terminus but is not sufficient for optimal transactivation. Alanine substitutions within the CBM reveal a close correlation between the transactivation and chromatin binding activities, implying a mechanistic link. In contrast to promoter activation, we find that the 223 amino acids of the LANA C terminus are sufficient to inhibit p53-mediated activation of the human BAX promoter, indicating that the CBM is not required for all transcription-related functions.