Biology subjects

Gurevich, V.

Publications and source records attributed to Gurevich, V..

10 recordsLinked to original sources

bioRxiv2026

KSHV upregulates the long non-coding RNA PURPL via p73 to suppress p53

Long non-coding RNAs (lncRNAs) are a class of regulatory RNAs with lengths exceeding 200 nucleotides. The lncRNA PURPL (p53 upregulated regulator of p53 levels) is a p53-regulated gene. PURPL is an oncogenic lncRNA that represses p53 function; it is upregulated in many tumors and promotes cancer cell proliferation. Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of all forms of Kaposi sarcoma (KS) and is tightly associated with primary effusion lymphoma (PEL). We found upregulation of PURPL in both de novo and chronically KSHV-infected cells. We found that p73 (a p53 homolog) associates with the PURPL promoter and upregulates PURPL expression. Previously, it has been shown that KSHV-encoded latency-associated nuclear antigen (LANA) stabilizes p73. We found that ectopic expression of LANA increased both p73 protein levels and PURPL expression. p73 knockdown abrogates PURPL upregulation following KSHV infection. Antisense oligonucleotides (ASO) targeting PURPL reduced PEL cell proliferation and increased acetylated p53 levels. In contrast to p53, p73 is not repressed by PURPL RNA. In summary, our study reveals how an oncogenic virus can utilize p73 to upregulate the oncogenic lncRNA PURPL as an additional way to repress p53. Moreover, we propose another mechanism by which p73 may promote tumor growth.

cancer biology↗

bioRxiv2026

LINE-1 transposable element is expressed in Kaposi's sarcoma and regulates gene expression in KSHV-infected cells

Long interspersed nuclear element-1 (LINE-1/L1) retrotransposons are expressed in various human cancers. Kaposis sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposis Sarcoma (KS) and Primary Effusion Lymphoma (PEL), which are characterized by global transcriptional reprogramming. Previously, we have found upregulation of L1 in PEL. Here, we show that knockdown of L1 in PEL cells significantly impaired their proliferation and induced broad transcriptional changes encompassing DNA replication, mitotic progression, and genome maintenance pathways. Analysis of KSHV-infected mouse and human mesenchymal stem cell models revealed that the KS-mimicking microenvironment robustly upregulates L1 expression, with sustained expression observed in KSHV-induced tumor cells even after the loss of the viral episome. Reanalysis of RNA-seq data from KS patient biopsies further showed elevated L1 expression in both endemic and AIDS-associated epidemic KS, with partial reduction following antiretroviral therapy. L1 ORF1p protein expression was confirmed by immunohistochemistry in KS tissues. Reanalysis of the enzymes that can modulate DNA methylation, a process that regulates L1 expression, revealed upregulation of DNMT3A and TET2 in both mouse KSHV tumor models and human KS. Altogether, our findings establish the L1 activation as a hallmark of KSHV-driven oncogenesis.

microbiology↗

bioRxiv2026

Arrestin-3 scaffolds multiple MAP3Ks driving stress-induced JNK3 activation and cell death.

Non-visual arrestin-3 (a.k.a. {beta}-arrestin-2) functions as a scaffold facilitating the activation of c-Jun N-terminal kinases (JNKs), an important pathway regulating cell fate. Here, we demonstrate that arrestin-3 scaffolds not only previously identified ASK1, but facilitates signaling by several MAP3Ks, including ZAK, ZAK{beta}, MEKK1, and TAK1. We identified ZAK (sterile alpha motif and leucine zipper-containing kinase) as the predominant MAP3K mediating arrestin-3-dependent JNK3 signaling and chemotherapy drug-induced cell death in HEK293 cells. We also showed that a 16-residue-long arrestin-3-derived peptide binds ZAK and fulfills the scaffolding function of full-length arrestin-3, sensitizing cells to death induced by chemotherapy drugs. These findings demonstrate that arrestin-3 is a versatile facilitator of stress signaling and suggest that functional peptide mimics can be used therapeutically to facilitate drug-induced death of cancer cells.

cell biology↗

bioRxiv2025

Cytoplasmic tail composition modulates the G protein and arrestin-3 signaling bias of the adhesion GPCR LPHN2

The class B2 Adhesion GPCRs (aGPCRs) combine cell adhesion with GPCR signaling to control diverse developmental and physiological processes. How aGPCRs interact with and integrate distinct groups of effectors including G proteins, arrestins, and G protein receptor kinases (GRKs) remains unclear. Here, we find that diversity in the aGPCR C-terminal intracellular tail modulates G protein activation, arrestin-3 recruitment, and utilization of selective GRKs in LPHN2, a postsynaptic aGPCR essential for synapse formation. The C-terminal tail of LPHN2 is required for G protein activation and arrestin-3 recruitment. LPHN2 with an intact tail recruits arrestin-3 in the absence of G protein activation, suggesting constitutive arrestin-3-biased signaling. Alternative splicing of the LPHN2 tail modulates G protein activation and arrestin-3 binding independently, supporting that it controls G protein vs arrestin-3 bias. GRKs are important but not essential for arrestin-3 recruitment to LPHN2. Moreover, GRK2 increases arrestin-3 recruitment only in a subset of LPHN2 variants. Collectively, these results show that the mechanisms of the interactions of class B2 aGPCRs and arrestin are distinct from those of class A GPCRs and that splicing of the LPHN2 C-terminal tail determines G protein vs arrestin bias.

molecular biology↗

bioRxiv2024

A small molecule enhances arrestin-3 binding to the β2-adrenergic receptor

G protein-coupled receptor (GPCR) signaling is terminated by arrestin binding to a phosphorylated receptor. Binding propensity has been shown to be modulated by stabilizing the pre-activated state of arrestin through point mutations or C-tail truncation. Here, we hypothesize that pre-activated rotated states can be stabilized by small molecules, and this can promote binding to phosphorylation-deficient receptors, which underly a variety of human disorders. We performed virtual screening on druggable pockets identified on pre-activated conformations in Molecular Dynamics trajectories of arrestin-3, and found a compound targeting an activation switch, the back loop at the inter-domain interface. According to our model, consistent with available biochemical and structural data, the compound destabilized the ionic lock between the finger and the back loop, and enabled transition of the gate loop towards the pre-activated state, which stabilizes pre-activated inter-domain rotation. The predicted binding pocket is consistent with saturation-transfer difference NMR data indicating close contact between the piperazine moiety of the compound and C/finger loops. The compound increases in-cell arrestin-3 binding to phosphorylation-deficient and wild-type {beta}2-adrenergic receptor, but not to muscarinic M2 receptor, as verified by FRET and NanoBiT. This study demonstrates that the back loop can be targeted to modulate interaction of arrestin with phosphorylation-deficient GPCRs in a receptor-specific manner.

cell biology↗

bioRxiv2024

KSHV genome harbors both constitutive and lytically induced enhancers

Kaposis sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements (CRE), such as enhancers and silencers, can regulate gene expression in a position and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream to the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency promoter (LANAp). Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron region, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the Terminal Repeats (TRs) acted as lytically induced enhancers. Expression of the Replication and Transcription Activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency protein LANA with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses. ImportanceIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, where one seems to be a tissue- specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the Replication and Transcription Activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and lytic genes locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers with their regulated genes. The terminal repeats spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency protein LANA with the activating effect of RTA to regulate latency to lytic transition.

microbiology↗

bioRxiv2023

Arrestin-3-assisted activation of JNK3 mediates dopaminergic behavioral and signaling plasticity in vivo.

In rodents with unilateral ablation of the substantia nigra neurons supplying dopamine to the striatum, chronic treatment with the dopamine precursor L-DOPA or dopamine agonists induces a progressive increase of behavioral responses, a process known as behavioral sensitization. The sensitization is blunted in arrestin-3 knockout mice. Using virus-mediated gene delivery to the dopamine-depleted striatum of arrestin-3 knockout mice, we found that the restoration of arrestin-3 fully rescued behavioral sensitization, whereas its mutant defective in JNK activation did not. A 25-residue arrestin-3-derived peptide that facilitates JNK3 activation in cells, expressed ubiquitously or selectively in the direct pathway striatal neurons, fully rescued sensitization, whereas an inactive homologous arrestin-2-derived peptide did not. Behavioral rescue was accompanied by the restoration of JNK3 activity and of JNK-dependent phosphorylation of the transcription factor c-Jun in the dopamine-depleted striatum. Thus, arrestin-3-dependent JNK3 activation in direct pathway neurons is a critical element of the molecular mechanism underlying sensitization.

neuroscience↗

bioRxiv2021

Unidirectional recruitment relations between proteins can be determined by a CRISPR/dCas9 recruitment assay

Kaposis sarcoma associated herpesvirus (KSHV, HHV-8) is associated with several human malignancies. During latency the viral genomes reside in the nucleus of infected cells as large non-integrated plasmids, known as episomes. All KSHV infected cells express LANA, and LANA is essential for viral latency. LANA binding to the viral episomes is critical for both replication of the viral genomes during latency, and for tethering the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes are critical for proper function of many nuclear processes. To test for recruitment directionality between LANA and cellular proteins we directed LANA via catalytically inactive Cas9 (dCas9) to a repeat sequence to obtain easily detectable dots. Then, recruitment of nuclear proteins to these dots can be evaluated. We found that LANA recruited its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1) which interacts with the N-terminal of MeCP2, was able to recruit MeCP2. Forcing MeCP2 dimerization via the tandem epitopes of SunTag, allows LANA to recruit MeCP2 in infected cells. We propose that available interacting domains force this recruitment directionality. Knock-down of MeCP2 by shRNA, as well as cells derived from Rett syndrome and express a mutant MeCP2 (T158M), dramatically reduced the ability of LANA to support viral latency. Therefore, this unidirectional recruitment of LANA by MeCP2 identified MeCP2 as a critical factor for KSHV viral maintenance. Significance StatementUsing a CRISPR/Cas9 recruitment assay, we show that some interacting proteins have a unidirectional recruitment property, where only one of the proteins can recruit its partner. We found unidirectional recruitment relations between the methylated DNA binding protein MeCP2 and KSHV encoded LANA. Where MeCP2 recruits LANA, but LANA is unable to recruit MeCP2. We were able to break this unidirectional recruitment by forcing MeCP2 dimerization. We propose that this unidirectional recruitment is the result of available interacting domains. Furthermore, this unidirectional recruitment seems to be critical for viral latency since LANA fails to maintain the viral genomes in MeCP2 mutant cells. Therefore, in this case unidirectional recruitment is a matter of survival or extinction.

microbiology↗

bioRxiv2019

Arginase Inhibition Supports Survival and Differentiation of Neuronal Precursors in Adult Alzheimer's Disease Mice

Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimers disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administer an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and apply an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidence a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline shows no significant effect upon the progenitor cell number and constitution. We demonstrate that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identify a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.

neuroscience↗

bioRxiv2018

L-Norvaline, a New Therapeutic Agent against Alzheimer s disease.

Alzheimers disease (AD) is a slowly progressive neurodegenerative disorder with an insidious onset. The disease is characterized by cognitive impairment and a distinct pathology with neuritic plaques and neurofibrillary tangles.\n\nGrowing evidence highlights the role of arginase activity in the manifestation of AD. Upregulation of arginase was shown to contribute to endothelial dysfunction, atherosclerosis, diabetes, and neurodegeneration. Regulation of arginase activity appears to be a promising approach for interfering with the pathogenesis of AD and other metabolic disorders. Therefore, the enzyme represents a novel therapeutic target.\n\nHere, we administer an arginase inhibitor L-norvaline to a mouse model of AD. Then, we evaluate the neuroprotective effect of L-norvaline using immunohistochemistry, proteomics, and quantitative polymerase chain reaction assays. Finally, we identify the biological pathways activated by the treatment.\n\nRemarkably, we find that L-norvaline treatment reverses the cognitive decline in AD mice. We show the treatment is neuroprotective as indicated by reduced beta-amyloidosis, alleviated microgliosis, and TNF transcription levels. Moreover, elevated levels of neuroplasticity related protein PSD-95 were detected in the hippocampi of mice treated with L-norvaline. Furthermore, we disclose several biological pathways, which are involved in cell survival and neuroplasticity and are activated by the treatment.\n\nThrough these modes of action, L-norvaline has the potential to improve the symptoms of AD and even interfere with its pathogenesis. As such, L-norvaline is a promising neuroprotective molecule that might be tailored for the treatment of a range of neurodegenerative disorders.

neuroscience↗