Rolando Cuevas

Physical and Functional Interaction between the Dopamine Transporter and the Synaptic Vesicle Protein Synaptogyrin-3

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein–protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Cullin 1 Functions as a Centrosomal Suppressor of Centriole Multiplication by Regulating Polo-like Kinase 4 Protein Levels

Abnormal centrosome and centriole numbers are frequently detected in tumor cells where they can contribute to mitotic aberrations that cause chromosome missegregation and aneuploidy. The molecular mechanisms of centriole overduplication in malignant cells, however, are poorly characterized. Here, we show that the core SKP1-cullin-F-box component cullin 1 (CUL1) localizes to maternal centrioles and that CUL1 is critical for suppressing centriole overduplication through multiplication, a recently discovered mechanism whereby multiple daughter centrioles form concurrently at single maternal centrioles. We found that this activity of CUL1 involves the degradation of Polo-like kinase 4 (PLK4) at maternal centrioles. PLK4 is required for centriole duplication and strongly stimulates centriole multiplication when aberrantly expressed. We found that CUL1 is critical for the degradation of active PLK4 following deregulation of cyclin E/cyclin-dependent kinase 2 activity, as is frequently observed in human cancer cells, as well as for baseline PLK4 protein stability. Collectively, our results suggest that CUL1 may function as a tumor suppressor by regulating PLK4 protein levels and thereby restraining excessive daughter centriole formation at maternal centrioles.

Centrosome Overduplication, Chromosomal Instability, and Human Papillomavirus Oncoproteins

Centrosome aberrations are a frequent finding in human tumors. However, very little is known about the molecular mechanisms leading to disruption of centrosome duplication control and the functional consequences of aberrant centrosome numbers. The high‐risk human papillomavirus Type 16 (HPV‐16) E6 and E7 oncoproteins are overexpressed in HPV‐associated malignancies of the anogenital tract and have been instrumental in delineating different pathways of centrosome amplification. Whereas the E6 oncoprotein was found to provoke centrosome accumulation, the HPV‐16 E7 oncoprotein triggers a genuine disruption of the centrosome duplication cycle. Importantly, the E7 oncoprotein can rapidly cause centrosome overduplication through a pathway that involves the concurrent formation of multiple daughters at single maternal centrioles (centriole flowers). Several lines of evidence suggest that cyclin E/CDK2 complexes and Polo‐like kinase 4 (PLK4) are crucial players in this process. These findings underscore that the HPV‐16 E7 oncoprotein is a unique tool to dissect normal and abnormal centriole biogenesis and the underlying molecular circuitry. Environ. Mol. Mutagen. 2009.

Daughter centriole elongation is controlled by proteolysis.

This report shows that inhibition of proteolysis can cause abnormal centriole elongation and identifies several centrosome proteins involved in this process by using a small interfering RNA screen.

FGF-2 Disrupts Mitotic Stability in Prostate Cancer through the Intracellular Trafficking Protein CEP57

Malignant tumors with deregulated FGF-2 expression such as prostate cancer are also frequently aneuploid. Aneuploidy can be caused by cell division errors due to extra centrosomes and mitotic spindle poles. However, a link between FGF-2 overexpression and chromosome missegregation has so far been elusive. Here, we show that FGF-2 rapidly uncouples centrosome duplication from the cell division cycle in prostate cancer cells through CEP57, an intracellular FGF-2-binding and trafficking factor. CEP57 was initially identified as a regulator of centriole overduplication in an RNA interference screen. We subsequently found that CEP57 rapidly stimulates centriole overduplication and mitotic defects when overexpressed and is required not only for FGF-2-induced centriole overduplication but also for normal centriole duplication. We provide evidence that CEP57 functions by modulating tubulin acetylation, thereby promoting daughter centriole stability. CEP57 was found to be overexpressed on the mRNA and protein level in a subset of prostate cancers, of which the vast majority also showed FGF-2 upregulation. Taken together, our results show an unexpected link between altered microenvironmental signaling cues such as FGF-2 overexpression and mitotic instability and provide a rationale for the therapeutic targeting of the FGF-2/FGFR1/CEP57 axis in prostate cancer. Cancer Res; 73(4); 1400-10. ©2012 AACR.

2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

ABSTRACT 2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.

Tripeptidyl Peptidase II Is Required for c-MYC–Induced Centriole Overduplication and a Novel Therapeutic Target in c-MYC–Associated Neoplasms

Centrosome aberrations are frequently detected in c-MYC-associated human malignancies. Here, we show that c-MYC-induced centrosome and centriole overduplication critically depend on the protease tripeptidyl peptidase II (TPPII). We found that TPPII localizes to centrosomes and that overexpression of TPPII, similar to c-MYC, can disrupt centriole duplication control and cause centriole multiplication, a process during which maternal centrioles nucleate the formation of more than a single daughter centriole. We report that inactivation of TPPII using chemical inhibitors or siRNA-mediated protein knockdown effectively reduced c-MYC-induced centriole overduplication. Remarkably, the potent and selective TPPII inhibitor butabindide not only potently suppressed centriole aberrations but also caused significant cell death and growth suppression in aggressive human Burkitt lymphoma cells with c-MYC overexpression. Taken together, these results highlight the role of TPPII in c-MYC-induced centriole overduplication and encourage further studies to explore TPPII as a novel antineoplastic drug target.

MOV10 Provides Antiviral Activity against RNA Viruses by Enhancing RIG-I–MAVS-Independent IFN Induction

Moloney leukemia virus 10, homolog (MOV10) is an IFN-inducible RNA helicase, associated with small RNA-induced silencing. In this article, we report that MOV10 exhibits antiviral activity, independent of its helicase function, against a number of positive- and negative-strand RNA viruses by enhancing type I IFN induction. Using a number of genome-edited knockout human cells, we show that IFN regulatory factor 3–mediated IFN induction and downstream IFN signaling through IFN receptor was necessary to inhibit virus replication by MOV10. MOV10 enhanced IFN regulatory factor 3–mediated transcription of IFN. However, this IFN induction by MOV10 was unique and independent of the known retinoic acid–inducible gene I/mitochondrial antiviral-signaling protein–mediated RNA-sensing pathway. Upon virus infection, MOV10 specifically required inhibitor of κB kinase ε, not TANK-binding kinase 1, for its antiviral activity. The important role of MOV10 in mediating antiviral signaling was further supported by the finding that viral proteases from picornavirus family specifically targeted MOV10 as a possible innate immune evasion mechanism. These results establish MOV10, an evolutionary conserved protein involved in RNA silencing, as an antiviral gene against RNA viruses that uses an retinoic acid–inducible gene I–like receptor–independent pathway to enhance IFN response.

Abstract 2974: Cep57 is a novel regulator of centriole overduplication in tumor cells

Abnormal multipolar mitoses are commonly observed in human cancers where they can increase the risk for chromosome segregation errors, numerical chromosomal instability and malignant progression. The mitotic spindle poles are formed by centrosomes and tumor cells frequently show abnormal centrosomes numbers. The molecular mechanisms leading to numerical centrosome aberrations are not understood in detail. The centrosome consists of two centrioles, which normally duplicate in synchrony with the cell division cycle. We recently identified a novel mechanisms leading to abnormal centriole numbers in tumor cells in which maternal centrioles generate more than the normal one daughter at the same time (centriole multiplication). Using a siRNA library screen enriched for centrosomal proteins, we identified Cep57 as a novel protein involved in centriole multiplication. Cep57 was found to localize to centrosomes and to co-localize and functionally interact with Polo-like kinase 4 (PLK4), a master regulator of centriole duplication control. Cep57 overexpression was found to promote abnormal centrosome and centriole numbers as well as mitotic abnormalities with a significant number of cells showing multipolar metaphases as well as multipolar anaphases and telophases. Cep57 maps to chromosome 11q21, a region that is amplified in several cancer types including hematological malignancies and prostate cancer. Taken together, we identified Cep57 as regulator of centriole duplication in tumor cells and a candidate oncogene. Experiments to target the Cep57-PLK4 axis with small molecules to prevent cell division errors and malignant progression are currently under way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2974.