SIP-428, a SIR2 Deacetylase Enzyme and Its Role in Biotic Stress Signaling Pathway

SABP2 (Salicylic Acid Binding Protein 2) plays a vital role in the salicylic acid signaling pathway of plants both regarding basal resistance and systemic acquired resistance against pathogen infection. SIP-428 (SABP2 Interacting Protein-428) is a Silent information regulator 2 (SIR2) like deacetylase enzyme that physically interacts with SABP2 in a yeast two-hybrid interaction and confirmed independently by a GST pull-down assay. We demonstrated that SIP- 428 is an NAD+ dependent SIR2 deacetylase enzyme. Transgenic tobacco plants silenced in SIP- 428 expression via RNAi showed enhanced basal resistance to microbial pathogens. Moreover, these SIP-428-silenced lines also exhibited a robust induction of systemic acquired resistance. In contrast, the transgenic tobacco lines overexpressing SIP-428 showed compromised basal resistance and failed to induce systemic acquired resistance. These results indicate that SIP-428 is likely a negative regulator of SA-mediated plant immunity. Experiments using a SABP2 inhibitor showed that SIP-428 likely functions upstream of SABP2 in the salicylic acid signaling pathway. It also indicates that SABP2 is dependent on SIP-428 for its role in the SA signaling pathway. Subcellular localization studies using confocal microscopy and subcellular fractionation showed that SIP-428 localized in the mitochondria. These results clearly show a role for SIP-428 in plant immunity

LncRNA HOTAIRM1 Promotes MDSC Expansion and Suppressive Functions Through the HOXA1-miR124 Axis During HCV Infection

HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that plays a pivotal role in regulating myeloid cell development via targeting HOXA1 gene expression. We and others have previously shown that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature myeloid cells, expand during chronic viral (HCV, HIV) infections. However, the role of HOTAIRM1 in the development and suppression of MDSCs during viral infection remains unknown. In this study, we demonstrate that the expressions of HOTAIRM1 and its target HOXA1 are substantially upregulated to promote the expressions of immunosuppressive molecules, including arginase 1, inducible nitric oxide synthase, signal transducer and activator of transcription 3, and reactive oxygen species, in CD33+ myeloid cells derived from hepatitis C virus (HCV)-infected patients. We show that HCV-associated exosomes (HCV-Exo) can modulate HOTAIRM1, HOXA1, and miR124 expressions to regulate MDSC development. Importantly, overexpression of HOTAIRM1 or HOXA1 in healthy CD33+ myeloid cells promoted the MDSC differentiation and suppressive functions; conversely, silencing of HOTAIRM1 or HOXA1 expression in MDSCs from HCV patients significantly reduced the MDSC frequency and their suppressive functions. In essence, these results indicate that the HOTAIRM1-HOXA1-miR124 axis enhances the differentiation and suppressive functions of MDSCs and may be a potential target for immunomodulation in conjunction with antiviral therapy during chronic viral infection

HCV-Associated Exosomes Upregulate RUNXOR and RUNX1 Expressions to Promote MDSC Expansion and Suppressive Functions through STAT3-miR124 Axis

RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA and plays a pivotal role in the differentiation of myeloid cells via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported that myeloid-derived suppressor cells (MDSCs) expand and inhibit host immune responses during chronic viral infections; however, the mechanisms responsible for MDSC differentiation and suppressive functions, in particular the role of RUNXOR-RUNX1, remain unclear. Here, we demonstrated that RUNXOR and RUNX1 expressions are significantly upregulated and associated with elevated levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS) in MDSCs during chronic hepatitis C virus (HCV) infection. Mechanistically, we discovered that HCV-associated exosomes (HCV-Exo) can induce the expressions of RUNXOR and RUNX1, which in turn regulates miR-124 expression via STAT3 signaling, thereby promoting MDSC differentiation and suppressive functions. Importantly, overexpression of RUNXOR in healthy CD33+ myeloid cells promoted differentiation and suppressive functions of MDSCs. Conversely, silencing RUNXOR or RUNX1 expression in HCV-derived CD33+ myeloid cells significantly inhibited their differentiation and expressions of suppressive molecules and improved the function of co-cultured autologous CD4 T cells. Taken together, these results indicate that the RUNXOR-RUNX1-STAT3-miR124 axis enhances the differentiation and suppressive functions of MDSCs and could be a potential target for immunomodulation in conjunction with antiviral therapy during chronic HCV infection

ATM Deficiency Accelerates DNA Damage, Telomere Erosion, and Premature T Cell Aging in HIV-Infected Individuals on Antiretroviral Therapy

HIV infection leads to a phenomenon of inflammaging, in which chronic inflammation induces an immune aged phenotype, even in individuals on combined antiretroviral therapy (cART) with undetectable viremia. In this study, we investigated T cell homeostasis and telomeric DNA damage and repair machineries in cART-controlled HIV patients at risk for inflammaging. We found a significant depletion of CD4 T cells, which was inversely correlated with the cell apoptosis in virus-suppressed HIV subjects compared to age-matched healthy subjects (HS). In addition, HIV CD4 T cells were prone to DNA damage that extended to chromosome ends—telomeres, leading to accelerated telomere erosion—a hallmark of cell senescence. Mechanistically, the DNA double-strand break (DSB) sensors MRE11, RAD50, and NBS1 (MRN complex) remained intact, but both expression and activity of the DNA damage checkpoint kinase ataxia-telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in HIV CD4 T cells. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in healthy CD4 T cells following ATM knockdown or exposure to the ATM inhibitor KU60019 in vitro, recapitulating the biological effects observed in HIV-derived CD4 T cells in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, apoptosis, and cellular dysfunction in HIV-derived CD4 T cells. These results demonstrate that failure of DSB repair due to ATM deficiency leads to increased DNA damage and renders CD4 T cells prone to senescence and apoptotic death, contributing to CD4 T cell depletion or dysfunction in cART-controlled, latent HIV infection

Telomeric Injury by KML001 in Human T Cells Induces Mitochondrial Dysfunction Through p53-PGC-1α Pathway

Telomere erosion and mitochondrial dysfunction are prominent features of aging cells with progressive declines of cellular functions. Whether telomere injury induces mitochondrial dysfunction in human T lymphocytes, the major component of adaptive host immunity against infection and malignancy, remains unclear. We have recently shown that disruption of telomere integrity by KML001, a telomere-targeting drug, induces T cell senescence and apoptosis via the telomeric DNA damage response (DDR). In this study, we used KML001 to further investigate the role and mechanism of telomere injury in mitochondrial dysregulation in aging T cells. We demonstrate that targeting telomeres by KML001 induces mitochondrial dysfunction, as evidenced by increased mitochondrial swelling and decreased mitochondrial membrane potential, oxidative phosphorylation, mitochondrial DNA content, mitochondrial respiration, oxygen consumption, glycolysis, and ATP energy production. Mechanistically, we found that the KML001-induced telomeric DDR activated p53 signaling, which in turn repressed the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and nuclear respiratory factor 1 (NRF-1), leading to T cell mitochondrial dysfunction. These results, forging a direct link between telomeric and mitochondrial biology, shed new light on the human T cell aging network, and demonstrate that the p53-PGC-1α-NRF-1 axis contributes to mitochondrial dysfunction in the setting of telomeric DDR. This study suggests that targeting this axis may offer an alternative, novel approach to prevent telomere damage-mediated mitochondrial and T cell dysfunctions to combat a wide range of immune aging-associated human diseases

Inhibition of Topoisomerase IIA (Top2α) Induces Telomeric DNA Damage and T Cell Dysfunction During Chronic Viral Infection

T cells play a critical role in controlling viral infection; however, the mechanisms regulating their responses remain incompletely understood. Here, we investigated the role of topoisomerase IIA (Top2α, an enzyme that is essential in resolving entangled DNA strands during replication) in telomeric DNA damage and T cell dysfunction during viral infection. We demonstrated that T cells derived from patients with chronic viral (HBV, HCV, and HIV) infection had lower Top2α protein levels and enzymatic activity, along with an accumulation of the Top2α cleavage complex (Top2cc) in genomic DNA. In addition, T cells from virally infected subjects with lower Top2α levels were vulnerable to Top2α inhibitor-induced cell apoptosis, indicating an important role for Top2α in preventing DNA topological disruption and cell death. Using Top2α inhibitor (ICRF193 or Etoposide)-treated primary T cells as a model, we demonstrated that disrupting the DNA topology promoted DNA damage and T cell apoptosis via Top2cc accumulation that is associated with protein-DNA breaks (PDB) at genomic DNA. Disruption of the DNA topology was likely due to diminished expression of tyrosyl-DNA phosphodiesterase 2 (TDP2), which was inhibited in T cells in vitro by Top2α inhibitor and in vivo by chronic viral infection. These results suggest that immune-evasive viruses (HBV, HCV, and HIV) can disrupt T cell DNA topology as a mechanism of dysregulating host immunity and establishing chronic infection. Thus, restoring the DNA topologic machinery may serve as a novel strategy to protect T cells from unwanted DNA damage and to maintain immune competence

A Matter of Life or Death: Productively Infected and Bystander CD4 T Cells in Early HIV Infection

CD4 T cell death or survival following initial HIV infection is crucial for the development of viral reservoirs and latent infection, making its evaluation critical in devising strategies for HIV cure. Here we infected primary CD4 T cells with a wild-type HIV-1 and investigated the death and survival mechanisms in productively infected and bystander cells during early HIV infection. We found that HIV-infected cells exhibited increased programmed cell death, such as apoptosis, pyroptosis, and ferroptosis, than uninfected cells. However, productively infected (p24+) cells and bystander (p24-) cells displayed different patterns of cell death due to differential expression of pro-/anti-apoptotic proteins and signaling molecules. Cell death was triggered by an aberrant DNA damage response (DDR), as evidenced by increases in γH2AX levels, which inversely correlated with telomere length and telomerase levels during HIV infection. Mechanistically, HIV-infected cells exhibited a gradual shortening of telomeres following infection. Notably, p24+ cells had longer telomeres compared to p24- cells, and telomere length positively correlated with the telomerase, pAKT, and pATM expressions in HIV-infected CD4 T cells. Importantly, blockade of viral entry attenuated the HIV-induced inhibition of telomerase, pAKT, and pATM as well as the associated telomere erosion and cell death. Moreover, ATM inhibition promoted survival of HIV-infected CD4 T cells, especially p24+ cells, and rescued telomerase and AKT activities by inhibiting cell activation, HIV infection, and DDR. These results indicate that productively infected and bystander CD4 T cells employ different mechanisms for their survival and death, suggesting a possible pro-survival, pro-reservoir mechanism during early HIV infection

SIR2 DEACETYLASE ENZYME AND ITS POSSIBLE ROLE IN PATHOGEN INFECTION

Silent Information Regulator 2 (SIR2) have a phylogenetically conserved catalytic domain from bacteria to humans. It catalyzes NAD+ dependent deacetylase activity post-translationally on acetylated lysine residues present in the protein. Because SIR2 are NAD+ dependent, its activity gets influenced by the change in the level of NAD+. SIR2 is responsible for calorie restriction and increased replicative yeast lifespan. It breakdown high energy bond in nicotinamide adenine dinucleotide (NAD), and the synthesis of O-acetyl-ADP-ribose which is a novel product. Lysine de/acetylation of histone molecule plays a significant role in chromatin dynamics in eukaryotes, but little is known in term of non-histone molecule modification by SIR2 enzyme especially in the case of the plant. SIP-428 is one of the SABP2 interacting protein (SIP) that exhibit SIR2 deacetylase activity. SABP2 is one of the essential components of salicylic acid (SA) signaling pathway that converts inactive methyl salicylate (MeSA) to active SA to induce local as well as SAR. AtSRT2, an Arabidopsis homolog of SIP-428 negatively regulate the basal resistance. Although catalytic domain is conserved, functional divergence has been reported in the case of SIR2 homologs. Presence of acetylated lysine residue in many cellular and organellar proteins implicated the possible physiological and metabolic role of SIP-428. Our result demonstrated SIP-428 exhibited NAD+ dependent deacetylase activity, but its lysine residue found to be acetylated, which raises the possibility of a post-translation regulatory mechanism that modulates the activity of SIP-428. SIP-428 have non-histone substrate, the negative regulator of basal resistance, and SAR. To understand better about the role of SIP-428 in plant physiology how it plays a vital role in SABP2 signaling pathway we will be using transgenic tobacco plant with altered expression of SIP-428 (Silence and inducible overexpression). Verified T3 generation of silence line and T2 generation of overexpression were created. These transgenic plant will be used to answer the possible link between SIP-428 and SABP2 in response to pathogen infection

Characterization of SIP428: a NAD+-Dependent Deacetylase Enzyme, in Abiotic Stress.

SABP2-interacting protein 428(SIP428) is a SIR2-type deacetylase, also called sirtuins. The SIP428 proteins belong to a family of NAD+-dependent deacetylase enzyme that was identified in tobacco. SABP2 is an important methyl esterase enzyme that catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) during the pathogenic challenge. Accumulation of SA induces systemic acquired resistance (SAR), a broad-spectrum defense mechanism in other uninfected distal parts of the plant. Sirtuins play diverse roles in DNA repair, apoptosis, and stress responses. Cellular proteins are known to undergo posttranslational modifications such as methylation, phosphorylation, and ubiquitination. A more recent addition to the list is acetylation. Protein acetylation is a reversible modification that plays role in regulating transcription, activation, and deactivation of certain pathways by transferring acetyl group to lysine residues. This change neutralizes the positive charge of the amino group thereby affecting the biological function of the affected proteins. Preliminary research has shown that SIP428 is a non-histone deacetylase. To understand better about the role of SIP-428 in plant physiology and how it plays a vital role in SABP2 signaling pathway we will be using transgenic tobacco plant in which the expression of SIP 428 has been silenced/knocked down