Khalid Amine Timani

TIP110/p110nrb/SART3/p110 regulation of hematopoiesis through CMYC

Intracellular factors are involved in and essential for hematopoiesis. HIV-1 Tat-interacting protein of 110 kDa (TIP110; p110(nrb)/SART3/p110) is an RNA-binding nuclear protein implicated in the regulation of HIV-1 gene and host gene transcription, pre-mRNA splicing, and cancer immunology. In the present study, we demonstrate a role for TIP110 in the regulation of hematopoiesis. TIP110 was expressed in human CD34(+) cells and decreased with differentiation of CD34(+) cells. TIP110 mRNA was also expressed in phenotyped mouse marrow hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Using TIP110 transgenic (TIP110(TG)) and haploinsufficient (TIP110(+/-)) mice, we found that increased TIP110 expression enhanced HPC numbers, survival, and cell cycling, whereas decreased TIP110 expression had the opposite effects. Moreover, TIP110(+/-) bone marrow HPCs responded more effectively, and TIP110(TG) HPCs less effectively, than those of wild-type control mice to recovery from the cell-cycle-active drug 5-fluorouracil (5-FU). Unexplained sex differences were noted in HSC competitive repopulating ability, but not HPC numbers, in TIP110(TG) mice. Intracellularly, TIP110 regulated CMYC and GATA2 expression at the transcriptional level, and TIP110 and CMYC reciprocally regulated the expression of each other. These results demonstrate a role for TIP110 in the regulation of hematopoiesis, effects that are likely linked to TIP110 regulation of CMYC.

Regulation of ubiquitin-proteasome system-mediated Tip110 protein degradation by USP15.

Tip110 is a nuclear protein and has been shown to function in tumor antigenicity, regulation of gene transcription, pre-mRNA splicing, stem cell proliferation and differentiation, and embryonic development. To characterize the in vivo functions of Tip110, a transgene cassette expressing human Tip110 protein (hTip110) was used to generate hTip110 transgenic (Tg) mice. Unexpectedly, only Tip110 mRNA but not Tip110 protein was expressed in Tg MEF and tissues. Treatment of Tg MEF with proteasome inhibitors led to detection of hTip110 protein, which prompted us to investigate the regulatory mechanisms of Tip110 degradation in mouse cells. We found that hTip110 was more sensitive to ubiquitin-proteasome system (UPS)-mediated protein degradation than mouse Tip110 (mTip110), likely resulting from more hTip110 ubiquitination. Using affinity chromatography and proteomics, we identified USP15, a deubiquitinating enzyme, to be associated with Tip110. Tip110 expression led to re-distribution of USP15 from the cytoplasm to the nucleus and complete co-localization of Tip110 with USP15 in the nucleus, whereas USP15 expression resulted in hTip110 deubiquitination. Interestingly, USP15 knockdown restored hTip110 protein expression in Tg MEF and USP15 expression had little effects. Taken together, these results provide insights into the regulatory mechanism of human Tip110 degradation by USP15.

Tip110 interacts with YB-1 and regulates each other’s function

BackgroundTip110 plays important roles in tumor immunobiology, pre-mRNA splicing, expression regulation of viral and host genes, and possibly protein turnover. It is clear that our understanding of Tip110 biological function remains incomplete.ResultsHerein, we employed an immunoaffinity-based enrichment approach combined with protein mass spectrometry and attempted to identify Tip110-interacting cellular proteins. A total of 13 major proteins were identified to be complexed with Tip110. Among them was Y-box binding protein 1 (YB-1). The interaction of Tip110 with YB-1 was further dissected and confirmed to be specific and involve the N-terminal of both Tip110 and YB-1 proteins. A HIV-1 LTR promoter-driven reporter gene assay and a CD44 minigene in vivo splicing assay were chosen to evaluate the functional relevance of the Tip110/YB-1 interaction. We showed that YB-1 potentiates the Tip110/Tat-mediated transactivation of the HIV-1 LTR promoter while Tip110 promotes the inclusion of the exon 5 in CD44 minigene alternative splicing.ConclusionsTip110 and YB-1 interact to form a complex and mutually regulate each other’s biological functions.

STAT3 and its phosphorylation are involved in HIV-1 Tat-induced transactivation of glial fibrillary acidic protein.

Human immunodeficiency virus type 1 (HIV-1) Tat protein is a major pathogenic factor in HIV-associated neurological diseases; it exhibits direct neurotoxicity and indirect astrocyte-mediated neurotoxicity. We have shown that Tat alone is capable of activating glial fibrillary acidic protein (GFAP) expression and inducing astrocytosis involving sequential activation of early growth response protein 1 (Egr-1) and p300. In this study, we determined the roles of signal transducer and activator of transcription 3 (STAT3) in Tat-induced GFAP transactivation. STAT3 expression and phosphorylation led to significant increases in GFAP transcription and protein expression. Tat expression was associated with increased STAT3 expression and phosphorylation in Tat-expressing astrocytes and HIV-infected astrocytes. GFAP, Egr-1 and p300 transcription and protein expression all showed positive response to STAT3 and its phosphorylation. Importantly, knockdown of STAT3 resulted in significant decreases in Tat-induced GFAP and Egr-1 transcription and protein expression. Taken together, these findings show that STAT3 is involved in and acts upstream of Egr1 and p300 in the Tat-induced GFAP transactivation cascade and suggest important roles of STAT3 in controlling astrocyte proliferation and activation in the HIV-infected central nervous system.

Tip110 Protein Binds to Unphosphorylated RNA Polymerase II and Promotes Its Phosphorylation and HIV-1 Long Terminal Repeat Transcription

Background: Tip110 synergizes with HIV-1 Tat protein and transactivates Tat-mediated HIV-1 LTR promoter, but the underlying mechanisms have not been understood. Results: Tip110 bound to unphosphorylated RNAPII and led to increased P-TEFb recruitment and RNAPII phosphorylation, enhancing Tat-mediated transcription elongation of the HIV-1 LTR. Conclusion: Tip110 is involved in transcription regulation of the HIV-1 LTR promoter. Significance: These findings provide additional insights into HIV-1 transcription. Transcription plays an important role in both HIV-1 gene expression and replication and mandates complicated but coordinated interactions between the host and virus. Our previous studies have shown that an HIV-1 Tat-interacting protein of 110 kDa, Tip110, binds to and enhances Tat function in Tat-mediated HIV-1 gene transcription and replication (Liu, Y., Li, J., Kim, B. O., Pace, B. S., and He, J. J. (2002) HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110. J. Biol. Chem. 277, 23854–23863). However, the underlying molecular mechanisms by which this takes place were not understood. In this study, we demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. In addition, we detected Tip110 at the HIV-1 long terminal repeat (LTR) promoter and found that Tip110 expression was associated with increased phosphorylation of serine 2 of the heptapeptide repeats within the RNAPII C-terminal domain and increased recruitment of positive transcription elongation factor b to the LTR promoter. Consistent with these findings, we showed that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. Taken together, these findings have provided additional and mechanistic evidence to support Tip110 function in HIV-1 transcription.

Tip110: Physical properties, primary structure, and biological functions.

HIV-1 Tat-interacting protein of 110kDa (Tip110), also referred to as squamous cell carcinoma antigen recognized by T cells 3 (Sart3), p110 or p110(nrb), was initially identified as a cDNA clone (KIAA0156) without annotated functions. Over the past twenty years, several functions have been attributed to this protein. The proposed biological functions include roles for Tip110 in pre-mRNA splicing, gene transcription, stem cell biology, and development. Dysregulation of Tip110 is also a contributing factor in the development of cancer and other human diseases. It is clear that our understanding of this protein is rapidly evolving. In this review, we aimed to provide a summary of all the existing literature on this gene/protein and its proposed biological functions.

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation.

Tip110/SART3 regulates IL-8 expression and predicts the clinical outcomes in melanoma

Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition. Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis. We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions. This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling. We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability. Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent. These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies.

Abstract 4557: Tip110-mediated regulation of NF-κB activity is key to tumorigenesis

HIV-1 Tat-interacting protein (Tip110), also known as squamous cell carcinoma antigen recognized by T cells 3 (SART3), is a multifaceted nuclear protein and has been shown to function in tumor antigenicity. Tip110 protein expression is very low in normal tissues and non-proliferating cells but becomes highly elevated in a number of malignant tumor cell lines and cancerous tissues, pointing to its possible role in tumorigenesis. Our previous studies have shown that Tip110 specifically interacted with oncogenic deubiquitinating enzyme, USP15, and that ectopic expression of the Tip110 protein led to re-distribution of USP15 from the cytoplasm to the nucleus. USP15 is known to inhibit TNF-α-induced NF-κB activity by modulation of IκB-α ubiquitination. In the current study, we demonstrated that Tip110 augmented TNF-α-induced transcription activity of NF-κB, and that deletion of the nuclear localization domain in Tip110 obliterated the observed enhancement of TNF-α-induced NF-κB activity, providing the very first evidence to support the specific role of Tip110 in NF-κB activation. In addition, we found that Tip110 expression modulated IκB-α phosphorylation and that activation of NF-κB by Tip110 was mediated through the p38/MAPK pathway. Moreover, we showed that ectopic expression of Tip110 induced translocation of NF-κB p65 from the cytoplasm to the nucleus, reminiscent of the aforementioned translocation of USP15, in the absence of direct interaction of Tip110 with either p65 or IκB-α. Taken together, these results indicate that opposing regulation of TNFα-induced NF-κB activity by Tip110 and USP15 could be pivotal for modulation of tumorigenesis. Further delineation of the underlying signaling cascades will likely provide new insights into development of anti-tumor agents. Citation Format: Khalid Amine Timani, Amanda Whitmill, Ying Liu, Johnny J. He. Tip110-mediated regulation of NF-κB activity is key to tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4557.