Shohreh Amini

Publications of Shohreh Amini

• Growth inhibition of malignant glioblastoma by DING protein.

  Authors: Markus J Bookland, Nune Darbinian, Michael Weaver, Shohreh Amini, Kamel Khalili

  Journal of neuro-oncology. 11/2011; 107(2):247-56.

  Malignant gliomas are a highly aggressive type of brain tumor with extremely poor prognosis. These tumors are highly invasive and are often surgically incurable and resistant to chemotherapeutics andMalignant gliomas are a highly aggressive type of brain tumor with extremely poor prognosis. These tumors are highly invasive and are often surgically incurable and resistant to chemotherapeutics and radiotherapy. Thus, novel therapies that target pathways involved in growth and survival of the tumor cells are required for the treatment of this class of brain tumors. Previous studies revealed that epidermal growth factor receptor and extracellular-signal-regulated kinases (ERKs), which are involved in the induction of cell proliferation, are activated in the most aggressive type of glioma, i.e. glioblastoma multiforme (GBM). In fact, GBMs with increased levels of ERK activity exhibit a more aggressive phenotype than the others with moderate ERK activity, pointing to the importance of ERK and its kinase activity in the development and progression of these tumors. In this study, we have evaluated the effect of p38SJ, a novel member of the DING family of proteins, derived from Hypericum perforatum calluses, on the growth of malignant glioma cell lines, T98G and U-87MG by focusing on cell cycle and signaling pathways controlled by phosphorylation of various regulatory proteins including ERK. p38SJ, which exhibits profound phosphatase activity, shows the capacity to affect the phosphorylation status of several important kinases modulating signaling pathways, and cell growth and proliferation. Our results demonstrate that p38SJ reduces glioma cell viability and arrests cell cycle progression at G0/G1. The observed growth inhibitory effect of p38SJ is likely mediated by the downregulation of several cell cycle gatekeeper proteins, including cyclin E, Cdc2, and E2F-1. These results suggest that p38SJ may serve as a potential candidate for development of a therapeutic agent for the direct treatment of malignant gliomas and/or as a potential radiosensitizer.

• JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons.

  Authors: Nana Merabova, Rafal Kaminski, Barbara Krynska, Shohreh Amini, Kamel Khalili, Armine Darbinyan

  Journal of cellular physiology. 10/2011;

  An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in humanAn indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP-2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein-producing CG-4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP-Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen-activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein-induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

• Insulin-like growth factor-I-forkhead box O transcription factor 3a counteracts high glucose/tumor necrosis factor-α-mediated neuronal damage: implications for human immunodeficiency virus encephalitis.

  Authors: Anna Wilk, Katarzyna Urbanska, Shuo Yang, Jin Ying Wang, Shohreh Amini, Luis Del Valle, Francesca Peruzzi, Leonard Meggs, Krzysztof Reiss

  Journal of neuroscience research. 02/2011; 89(2):183-98.

  In HIV patients, antiretroviral medications trigger metabolic abnormalities, including insulin resistance. In addition, the inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated inIn HIV patients, antiretroviral medications trigger metabolic abnormalities, including insulin resistance. In addition, the inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated in human immunodeficiency virus encephalitis (HIVE), also induces insulin resistance and inflicts neuronal damage in vitro. In differentiated PC12 cells and rat cortical neurons, high glucose (HG; 25 mM) triggers reactive oxygen species (ROS) accumulation, contributing to the retraction of neuronal processes, with only a minimal involvement of neuronal apoptosis. In the presence of TNFα, HG-treated neurons undergo massive apoptosis. Because mammalian homolog of the Forkhead family of transcription factors, Forkhead box O transcription factor 3a (FOXO3a), controls ROS metabolism, we asked whether FOXO3a could affect the fate of differentiated neurons in the paradigm of HIVE. We observed FOXO3a nuclear translocation in HG-treated neuronal cultures, accompanied by partial loss of mitochondrial potential and gradual retraction of neuronal processes. Addition of TNFα to HG-treated neurons increased expression of the FOXO-dependent proapoptotic gene Bim, which resulted in extensive apoptotic death. Insulin-like growth factor-I (IGF-I) significantly lowered intracellular ROS, which was accompanied by IGF-I-mediated FOXO3a nuclear export and decrease in its transcriptional activity. The clinical relevance of these findings is supported by detection of nuclear FOXO3a in TUNEL-positive cortical neurons from HIVE, especially in brain areas characterized by elevated TNFα.

• Suppression of HIV-1 transcriptional elongation by a DING phosphatase.

  Authors: Nune Darbinian, Rebeccah Gomberg, Loriann Mullen, Samantha Garcia, Martyn K White, Kamel Khalili, Shohreh Amini

  Journal of cellular biochemistry. 01/2011; 112(1):225-32.

  HIV-1 gene transcription is controlled by the cooperation of viral and host factors which bind to specific DNA sequences within the viral promoter spanning the long terminal repeat (LTR). PreviouslyHIV-1 gene transcription is controlled by the cooperation of viral and host factors which bind to specific DNA sequences within the viral promoter spanning the long terminal repeat (LTR). Previously we showed that the St. John's Wort DING phosphatase, p27SJ, suppresses HIV-1 gene transcription by binding to the viral protein Tat and preventing its nuclear import. Here, we describe the inhibitory effect of p27SJ on the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). This inhibition leads to the suppression of the association of RNAPII with the LTR. Inhibition of binding of RNAPII to LTR by p27SJ resulted in the suppression of LTR transcription elongation and a decrease in LTR transcriptional activity. Another form of the St. John's Wort DING phosphatase, p38SJ, also suppressed binding of RNAPII to the LTR, reduced transcription elongation and was even more powerful than p27SJ in inhibiting the transcriptional activity of the LTR. Our data suggest a possible mechanism by which the p27SJ/p38SJ DING phosphatase can regulate HIV-1 LTR expression by inhibiting phosphorylation of the CTD of RNAPII and suppressing LTR transcription elongation.

• Regulation of the HIV-1 promoter by HIF-1α and Vpr proteins.

  Authors: Satish L Deshmane, Shohreh Amini, Satarupa Sen, Kamel Khalili, Bassel E Sawaya

  Virology journal. 01/2011; 8:477.

  We previously demonstrated the ability of HIV-1 Vpr protein to activate the oxidative stress pathway, thus leading to the induction of the hypoxia inducible factor 1 alpha (HIF-1α). Therefore, weWe previously demonstrated the ability of HIV-1 Vpr protein to activate the oxidative stress pathway, thus leading to the induction of the hypoxia inducible factor 1 alpha (HIF-1α). Therefore, we sought to examine the interplay between the two proteins and the impact of HIF-1α activation on HIV-1 transcription. Using transient transfection assays, we identified the optimal concentration of HIF-1α necessary for the activation of the HIV-1 promoter as well as the domain within HIF-1α responsible for this activation. Our findings indicated that activation of the HIV-1 LTR by Vpr is HIF-1α dependent. Furthermore, we showed that both Vpr and HIF-1α activate the HIV-1 promoter through the GC-rich binding domain within the LTR. Taken together, these data shed more light on the mechanisms used by Vpr to activate the HIV-1 promoter and placed HIF-1α as a major participant in this activation.

• Role of Purα in the cellular response to ultraviolet-C radiation.

  Authors: Rafal Kaminski, Laurelle Cheeseboro, Shohreh Amini, Edward M Johnson, Martyn K White, Kamel Khalili, Armine Darbinyan

  Cell cycle (Georgetown, Tex.). 10/2010; 9(20):4164-73.

  Purα is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Purα inPurα is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Purα in Ultraviolet-C (UVC) radiation-induced DNA damage and nucleotide excision repair (NER). Mouse embryo fibroblasts from PURA(-/-) knockout mice, which lack Purα, showed enhanced sensitivity to UVC irradiation as assessed by assays for cell viability and clonogenicity compared to Purα positive control cultures. In reporter plasmid reactivation assays to measure the removal of DNA adducts induced in vitro by UVC, the Purα-negative cells were less efficient in DNA damage repair. Purα-negative cells were also more sensitive to UVC-induced DNA damage measured by Comet assay and showed a decreased ability to remove UVC-induced cyclobutane pyrimidine dimers. In wild-type mouse fibroblasts, expression of Purα is induced following S-phase checkpoint activation by UVC in a similar manner to the NER factor TFIIH. Moreover, co-immunoprecipitation experiments showed that Purα physically associates with TFIIH. Thus, Purα has a role in NER and the repair of UVC-induced DNA damage.

• Activation of HIV-1 LTR by Rad51 in microglial cells.

  Authors: Inna Rom, Armine Darbinyan, Martyn K White, Jay Rappaport, Bassel E Sawaya, Shohreh Amini, Kamel Khalili

  Cell cycle (Georgetown, Tex.). 09/2010; 9(18):3715-22.

  Infection with HIV-1 induces a variety of biological alterations to the host that are beneficial to the life cycle of the virus but may have adverse effects on the host cell. Here we demonstrate thatInfection with HIV-1 induces a variety of biological alterations to the host that are beneficial to the life cycle of the virus but may have adverse effects on the host cell. Here we demonstrate that expression of Rad51, a major component of the homologous recombination-directed DNA repair (HRR) pathway, is induced upon HIV-1 infection of microglial cells. Activation of Rad51 expression positively impacts on HIV-1 LTR transcription through a region of the viral promoter known for binding the inducible transcription factor NFκB. Rad51 showed the ability to form a complex with the p65 subunit of NFκB and regulate the level of p65 interaction with LTR DNA encompassing the κB motif. This study provides evidence for reciprocal interaction of HIV-1 and a host DNA repair protein that impacts on expression of the viral genome. These results also point to the ability of HIV-1 to recruit proteins involved in DNA repair that are necessary for retroviral DNA integration, efficient replication and prevention of viral-induced cell death.

• p38SJ, a novel DINGG protein protects neuronal cells from alcohol induced injury and death.

  Authors: Shohreh Amini, Nana Merabova, Kamel Khalili, Nune Darbinian

  Journal of cellular physiology. 10/2009;

  Ethanol induces neuronal cell injury and death by dysregulating several signaling events that are controlled, in part, by activation of MAPK/ERK1/2 and/or inactivation of its correspondingEthanol induces neuronal cell injury and death by dysregulating several signaling events that are controlled, in part, by activation of MAPK/ERK1/2 and/or inactivation of its corresponding phosphatase, PP1. Recently, we have purified a novel protein of 38 kDa in size, p38SJ, from a callus culture of Hypericum perforatum, which belongs to an emerging DINGG family of proteins with phosphate binding activity. Here, we show that treatment of neuronal cells with p38SJ protects cells against injury induced by exposure to ethanol. Furthermore, pre-treatment of neuronal cells with p38SJ diminishes the level of the pro-apoptotic protein Bax and some events associated with apoptosis such as caspase 3 cleavage. In addition, by inducing stress, alcohol can elevate production of reactive oxygen species (ROS) that leads to a decrease in the activity of superoxide dismutase (SOD). Our results showed that p38SJ restores the activity of SOD in the ethanol treated neuronal cells. These observations provide a novel biological tool for developing new approaches for preventing neuronal cell death induced by ethanol and possibly treatment of neurological disorders associated with alcohol abuse. J. Cell. Physiol. (c) 2009 Wiley-Liss, Inc.

• Monocyte chemoattractant protein-1 (MCP-1): an overview.

  Authors: Satish L Deshmane, Sergey Kremlev, Shohreh Amini, Bassel E Sawaya

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 07/2009; 29(6):313-26.

  Chemokines constitute a family of chemoattractant cytokines and are subdivided into four families on the basis of the number and spacing of the conserved cysteine residues in the N-terminus of theChemokines constitute a family of chemoattractant cytokines and are subdivided into four families on the basis of the number and spacing of the conserved cysteine residues in the N-terminus of the protein. Chemokines play a major role in selectively recruiting monocytes, neutrophils, and lymphocytes, as well as in inducing chemotaxis through the activation of G-protein-coupled receptors. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages. Both CCL2 and its receptor CCR2 have been demonstrated to be induced and involved in various diseases. Migration of monocytes from the blood stream across the vascular endothelium is required for routine immunological surveillance of tissues, as well as in response to inflammation. This review will discuss these biological processes and the structure and function of CCL2.

• Evidence for phosphatase activity of p27SJ and its impact on the cell cycle.

  Authors: Nune Darbinian, Marta Czernik, Armine Darbinyan, Mikael Elias, Eric Chabriere, Surekha Bonasu, Kamel Khalili, Shohreh Amini

  Journal of cellular biochemistry. 05/2009;

  p27SJ, a novel protein isolated from St John's wort (Hypericum perforatum), belongs to an emerging family of DING proteins that are related to a prokaryotic phosphate-binding protein superfamily.p27SJ, a novel protein isolated from St John's wort (Hypericum perforatum), belongs to an emerging family of DING proteins that are related to a prokaryotic phosphate-binding protein superfamily. Here we demonstrate that p27SJ exhibits phosphatase activity and that its expression in cells decreases the level of phosphorylated Erk1/2, a key protein of several signaling pathways. Treatment of p27SJ-expressing cells with phosphatase inhibitors including okadaic acid, maintained Erk1/2 in its phosphorylated form, suggesting that dephosphorylation of Erk1/2 is mediated by p27SJ. Further, expression of p27SJ affects Erk1/2 downstream regulatory targets such as STAT3 and CREB. Moreover, the level of expression of cyclin A that associates with active ERK1/2 and is regulated by CREB, was modestly reduced in p27SJ-expressing cells. Accordingly, results from in vitro kinase assays revealed a noticeable decrease in the activity of cyclin A in cells expressing p27SJ. Cell cycle analysis demonstrated dysregulation at S and G2/M phases in cells expressing p27SJ, supporting the notion that a decline in cyclin A activity by p27SJ has a biological impact on cell growth. These observations provide evidence that p27SJ alters the state of Erk1/2 phosphorylation, and impacts several biological events associated with cell growth and function. J. Cell. Biochem. (c) 2009 Wiley-Liss, Inc.

• HIV-1 Vpr deregulates calcium secretion in neural cells.

  Authors: Inna Rom, Satish L Deshmane, Ruma Mukerjee, Kamel Khalili, Shohreh Amini, Bassel E Sawaya

  Brain research. 04/2009;

  The lack of productive infection of neurons by HIV-1 suggests that the neuronal damage seen in AIDS patients with cognitive disorders is caused indirectly via viral and cellular proteins withThe lack of productive infection of neurons by HIV-1 suggests that the neuronal damage seen in AIDS patients with cognitive disorders is caused indirectly via viral and cellular proteins with neurotoxic activity. Among HIV-1 proteins, Vpr has been shown to deregulate expression of various important cytokines and inflammatory proteins in infected and uninfected cells. However, the mechanisms underlying these changes remain unclear. Here, we demonstrate that neurons can take up Vpr that is released into the supernatant of HIV-infected microglia. We also found that administration of recombinant Vpr (rVpr) to human neurons resulted in a slow but sustained elevation of intracellular calcium [Ca(2+)]i. Interestingly, our data also show that [Ca(2+)]i elevation by Vpr leads to ROS production and impairs glutamate signaling in neuronal cells. Vpr disturbs calcium homeostasis through downregulation of endogenous PMCA. Finally, we found that the permeability of the plasma membrane increases in neurons treated with Vpr. Therefore, we conclude that soluble Vpr is a major viral factor that causes a disturbance in neuronal communication leading to neuronal dysfunction. The outcome of these studies will advance the understanding of HIV-1 pathogenesis and will help in the development of new therapeutic approaches.

• Activation of the oxidative stress pathway by HIV-1 Vpr leads to induction of hypoxia inducible factor 1 alpha expression.

  Authors: Satish L Deshmane, Ruma Mukerjee, Shongshan Fan, Luis Del Valle, Carine Michiels, Thersa Sweet, Inna Rom, Kamel Khalili, Jay Rappaport, Shohreh Amini, Bassel E Sawaya

  The Journal of biological chemistry. 03/2009;

  The detection of biomarkers of oxidative stress in brain tissue and cerebrospinal fluid of patients with HIV-1 dementia indicates the involvement of stress pathways in the neuropathogenesis of AIDS.The detection of biomarkers of oxidative stress in brain tissue and cerebrospinal fluid of patients with HIV-1 dementia indicates the involvement of stress pathways in the neuropathogenesis of AIDS. While the biological importance of oxidative stress on events involved in AIDS neuropathogenesis and the HIV-1 proteins responsible for oxidative stress remain to be elucidated, our results point to the activation of hypoxia-inducible factor 1 (HIF-1) upon HIV-1 infection and its elevation in brain cells of AIDS patients with dementia. HIF-1 is a transcription factor that is responsive to oxygen. Under hypoxic conditions, HIF-1a becomes stable and translocates to the nucleus where it dimerizes with ARNT and modulates gene transcription. Activation of HIF-1 can also be mediated by the HIV-1 accessory protein, Vpr. In addition, cellular events including reactive oxygen species (ROS) contribute to the induction of HIF-1a. Our results show that Vpr induces ROS by increasing H2O2 production, which can contribute to HIF-1a accumulation. Interestingly, increased levels of HIF-1a stimulated HIV-1 gene transcription through HIF-1 association with HIV-1 LTR DNA. These observations point to the existence of a positive feedback interplay between HIF-1a and Vpr and that, by inducing oxidative stress via activation of HIF-1, Vpr can induce HIV-1 gene expression and desregulate multiple host cellular pathways.

• IGF-IR in neuroprotection and brain tumors.

  Authors: Elisa Gualco, Jin Ying Wang, Luis Del Valle, Katarzyna Urbanska, Francesca Peruzzi, Kamel Khalili, Shohreh Amini, Krzysztof Reiss

  Frontiers in bioscience : a journal and virtual library. 02/2009; 14:352-75.

  The IGF-IR is a multifunctional tyrosine kinase receptor involved in several biological processes including cell proliferation, differentiation, DNA repair, and cell survival. In the brain IGF-IThe IGF-IR is a multifunctional tyrosine kinase receptor involved in several biological processes including cell proliferation, differentiation, DNA repair, and cell survival. In the brain IGF-I plays a critical role during embryonic and early postnatal development. In the mature brain, IGF-I binding sites have been found in different regions of the brain, and multiple reports confirmed a strong neuroprotective action of the IGF-IR against different pro-apoptotic insults. When the IGF-IR signaling system is insufficiently deployed, either by low level of expression in elderly individuals, or by the inhibition associated with inflammatory cytokines, neuronal function and survival could be compromised. The examples of such CNS pathologies include HIV associated dementia, diabetic neuropathies, and Alzheimer's disease. On the other hand, elevated expression activity of the IGF-IR may support uncontrolled cell proliferation and protection from apoptosis. Probably the best example of the IGF-IR involvement in brain tumors is medulloblastomas in which functional cooperation between viral oncoprotein, JC virus large T-antigen, and IGF-IR has been recently established. Therefore, better understanding of the beneficial and potentially harmful aspects of the IGF-IR can be critical for the development of new clinical regimens against neurodegenerative disorders and brain tumors.

• Evidence for Activation of the TGF-beta1 Promoter by C/EBPbeta and its Modulation by Smads.

  Authors: Selvajothi Abraham, Thersa Sweet, Kamel Khalili, Bassel E Sawaya, Shohreh Amini

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 12/2008;

  The transforming growth factor-beta1 (TGF-beta1) is a cytokine involved in many biological events inlcuding immunosuppression, angiogenesis, cell growth, and apoptosis. Expression of TGF-beta1 at theThe transforming growth factor-beta1 (TGF-beta1) is a cytokine involved in many biological events inlcuding immunosuppression, angiogenesis, cell growth, and apoptosis. Expression of TGF-beta1 at the transcriptional level is controlled by a series of ubiquitous and specialized factors whose activities can be modulated by a variety of signaling events. Here we demonstrate that activity of the TGF-beta1 promoter is increased by C/EBPbeta, a DNA-binding transcription factor whose activity can be influenced by several immunomodulators, in astrocytes and microglial cells. Interestingly, expression of Smad3 and Smad4, the downstream regulators of the TGF-beta1-signaling pathway, impairs the activity of C/EBPbeta on the TGF-beta1 promoter. Further, we demonstrate that MH2, a common domain among Smads that has protein-binding activities, interacts with C/EBPbeta and decreases its association with a region of the TGF-beta1 promoter that is responsive to C/EBPbeta activation. Interestingly, the p65 subunit of nuclear factor-kappaB (NF-kappaB), which also interacts with C/EBPbeta, cooperates with MH2 and decreased DNA-binding and transcriptional activities of C/EBPbeta on the TGF-beta1 promoter. These observations indicate that an autoregulatory mechanism, involving the MH2 domain of Smads, modulates activation of the TGF-beta1 promoter by C/EBPbeta. Further, our results show that the interplay between NF-kappaB and C/EBPbeta has an impact on the ability of C/EBPbeta to stimulate TGF-beta1 transcription, hence, suggesting that the cross-communication of signaling pathways that modulate NF-kappaB and C/EBPbeta may dictate the level of TGF-beta1 promoter activity.

• Involvement of the p53 and p73 transcription factors in neuroAIDS.

  Authors: Ruma Mukerjee, Satish L Deshmane, Shongshan Fan, Luis Del Valle, Martyn K White, Kamel Khalili, Shohreh Amini, Bassel E Sawaya

  Cell cycle (Georgetown, Tex.). 10/2008; 7(17):2682-90.

  HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it isHIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.

• Negative Regulation of AbetaPP Gene Expression by Pur-alpha.

  Authors: Nune Darbinian, Jianqi Cui, Anna Basile, Luis Del Valle, Jessica Otte, Judith Miklossy, Bassel E Sawaya, Shohreh Amini, Kamel Khalili, Jennifer Gordon

  Journal of Alzheimer's disease : JAD. 10/2008; 15(1):71-82.

  The nucleic acid binding protein, Pur-alpha, is best characterized as a transcription factor with affinity to single stranded G/C rich regions. Pur-alpha exhibits developmental and tissue-specificThe nucleic acid binding protein, Pur-alpha, is best characterized as a transcription factor with affinity to single stranded G/C rich regions. Pur-alpha exhibits developmental and tissue-specific regulation and plays a critical role in neuronal development and differentiation. Similar to Pur-alpha, the amyloid-beta protein precursor (AbetaPP) is a developmentally regulated protein which promotes neuronal survival. Both the human and mouse AbetaPP promoters contain multiple G/C rich sequences which regulate AbetaPP at the transcriptional and translational levels. Using an in vitro reporter assay, we confirmed that Pur-alpha consensus binding sites within the human AbetaPP promoter down-regulate AbetaPP transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays (ChIP) showed direct binding of Pur-alpha to the AbetaPP promoter. Down regulation of AbetaPP went beyond the transcriptional level as overexpression of Pur-alpha in glial and fibroblast cell lines decreased basal levels of AbetaPP while siRNA targeting Pur-alpha increased basal levels of AbetaPP. Similar findings were observed in brain tissue and fibroblasts from mice with targeted deletion of Pur-alpha. These data point to a novel mechanism of controlling AbetaPP levels by the transcriptional regulatory protein, Pur-alpha, and suggest that Pur-alpha may be involved in the dysregulation of AbetaPP in Alzheimer's disease.

• Creation of a bi-directional protein transduction system for suppression of HIV-1 expression by p27SJ.

  Authors: Nune Darbinian, Yuri Popov, Kamel Khalili, Shohreh Amini

  Antiviral research. 09/2008; 79(2):136-41.

  p27SJ is a novel protein from a callus culture of St. John's wort that modulates transcription of the HIV-1 promoter in several mammalian cells [Darbinian-Sarkissian, N., Darbinyan, A., Otte, J.,p27SJ is a novel protein from a callus culture of St. John's wort that modulates transcription of the HIV-1 promoter in several mammalian cells [Darbinian-Sarkissian, N., Darbinyan, A., Otte, J., Radhakrishnan, S., Sawaya, B.E., Arzumanyan, A., Chipitsyna, G., Popov, Y., Rappaport, J., Amini, S., Khalili, K., 2006. p27(SJ), a novel protein from St. John's wort, that suppresses expression of HIV-1 genome. Gene Ther. 13, 288-295]. Here, we armed p27SJ with signals from Ig-kappa light chain that allow its efficient excretion from the cells, and from HIV-1 Tat that facilitates its uptake by other cells for its utilization by a protein transduction method. We demonstrate that treatment of cells containing the HIV-1 LTR with conditioned media from cells expressing the armed p27SJ ((exc)p27SJ(upt)) results in suppression of the viral activation by the C/EBPbeta transcription factor. Once imported into the cells, (exc)p27SJ(upt) impacts the nuclear localization of C/EBPbeta and by retaining the protein in the cytoplasm affects its DNA binding and hence transcriptional activity. The armed p27SJ also inhibits Tat-induced activation of the LTR and decreases the level of viral replication in promonocytic cells including U-937 and T-lymphocytic cells. Our observations introduce a new bi-directional protein transduction system with a broad spectrum of applications for manufacturing therapeutic peptides by a specific group of cells called donor, and delivery to the target cells named recipient. Furthermore, our results support the utility of soluble p27SJ in suppressing transcription and replication of HIV-1 by interfering with the function of cellular proteins such as C/EBPbeta and viral activators including Tat.

• St. John's Wort protein, p27SJ, regulates the MCP-1 promoter.

  Authors: Ruma Mukerjee, Satish L Deshmane, Nune Darbinian, Marta Czernik, Kamel Khalili, Shohreh Amini, Bassel E Sawaya

  Molecular immunology. 09/2008; 45(15):4028-35.

  St. John's Wort is commonly known for its antiviral, antidepressant, and cytotoxic properties, but traditionally St. John's Wort has also been used to treat inflammation. In this study, we sought toSt. John's Wort is commonly known for its antiviral, antidepressant, and cytotoxic properties, but traditionally St. John's Wort has also been used to treat inflammation. In this study, we sought to characterize the mechanisms used by St. John's Wort to treat inflammation by examining the effect of the recently isolated protein from St. John's Wort, p27SJ on the expression of MCP-1. By employing an adenovirus expression vector, we demonstrate that a low concentration of p27SJ upregulates the MCP-1 promoter through the transcription factor C/EBPbeta. In addition, we found that C/EBPbeta-homologous protein (CHOP) or siRNA-C/EBPbeta significantly reduced the ability of p27SJ to activate MCP-1 gene expression. Results from protein-protein interaction studies illustrate the existence of a physical interaction between p27SJ and C/EBPbeta in microglial cells. The use of chromatin immunoprecipitation assay (ChIP) led to the identification of a new cis-element that is responsive to C/EBPbeta within the MCP-1 promoter. Association of C/EBPbeta with MCP-1 DNA was not affected by the presence of p27SJ. The biological activity of MCP-1 produced by cultures of adenovirus-p27SJ transduced cells was increased relative to controls as measured by the transmigration of human Jurkat cells. Thus, we conclude that at high concentration, p27SJ is a potential agent that may be developed as a modulator of MCP-1 leading to the inhibition of the cytokine-mediated inflammatory responses.

• HIV-1 Tat inhibits NGF-induced Egr-1 transcriptional activity and consequent p35 expression in neural cells.

  Authors: Nune Darbinian, Armine Darbinyan, Marta Czernik, Francesca Peruzzi, Kamel Khalili, Krzysztof Reiss, Jennifer Gordon, Shohreh Amini

  Journal of cellular physiology. 08/2008; 216(1):128-34.

  Infection with HIV-1 causes degeneration of neurons leading to motor and cognitive dysfunction in AIDS patients. One of the key viral regulatory proteins, Tat, which is released by infected cells,Infection with HIV-1 causes degeneration of neurons leading to motor and cognitive dysfunction in AIDS patients. One of the key viral regulatory proteins, Tat, which is released by infected cells, can be taken up by various uninfected cells including neurons and by dysregulating several biological events induces cell injury and death. In earlier studies, we demonstrated that treatment of neuronal cells with Tat affects the nerve growth factor (NGF) signaling pathway involving MAPK/ERK. Here we demonstrate that a decrease in the level of Egr-1, one of the targets for MAPK, by Tat has a negative impact on the level of p35 expression in NGF-treated neural cells. Further, we demonstrate a reduced level of Egr-1 association with the p35 promoter sequence in NGF-treated cells expressing Tat. As p35, by associating with Cdk5, phosphorylates several neuronal proteins including neurofilaments and plays a role in neuronal differentiation and survival, we examined kinase activity of p35 complexes obtained from cells expressing Tat. Results from H1 kinase assays showed reduced activity of the p35 complex from Tat-expressing cells in comparison to that from control cells. Accordingly, the level of phosphorylated neurofilaments was diminished in Tat-expressing cells. Similarly, treatment of PC12 cells with Tat protein or supernatant from HIV-1 infected cells decreased kinase activity of p35 in these cells. These observations ascribe a role for Tat in altering p35 expression and its activity that affects phosphorylation of proteins involved in neuronal cell survival.

• Development of a Bidirectional Caspase-3 Expression System for the Induction of Apoptosis.

  Authors: Martyn White, Shohreh Amini, Kamel Khalili, Nune Darbinian

  Cancer biology & therapy. 04/2008; 7(6).

  Caspase-3 is the executioner caspase of apoptosis whose activation in mammalian cells represents the last stage of the programmed cell death signaling pathway and the initiation of the lethalCaspase-3 is the executioner caspase of apoptosis whose activation in mammalian cells represents the last stage of the programmed cell death signaling pathway and the initiation of the lethal digestion of cell proteins. Active caspase-3 is a tetramer composed of two p12 and two p17 subunits derived from cleavage of procaspase-3 during activation. Here, we armed GFP-fusion proteins of both the caspase-3 p12 and p17 subunits with signals from Ig-kappa light chain that allows its efficient secretion from the cells (Sec) and from HIV-1 Tat that facilitates its uptake and nuclear translocation by other cells (NLS). We found that treatment of cells with conditioned media from cells expressing both Sec-GFP-p17-NLS and Sec-GFP-p12-NLS was able to transduce active caspase-3 with consequent cell death of treated cultures. Use of various combinations of constructs demonstrated that both subunits were required and that each one needed to possess both Sec and NLS. Our observations introduce a bidirectional protein transduction system with the ability to introduce active caspase-3 into cells and cause apoptosis. This system may have important therapeutic applications.