Publications by authors named "Belinda Z Leal"

9 Publications

  • Page 1 of 1

Human polyhomeotic homolog 3 (PHC3) sterile alpha motif (SAM) linker allows open-ended polymerization of PHC3 SAM.

Biochemistry 2012 Jul 28;51(27):5379-86. Epub 2012 Jun 28.

Department of Biochemistry and CTRC, University of Texas Health Science Center at San Antonio, 78229-3990, United States.

Sterile alpha motifs (SAMs) are frequently found in eukaryotic genomes. An intriguing property of many SAMs is their ability to self-associate, forming an open-ended polymer structure whose formation has been shown to be essential for the function of the protein. What remains largely unresolved is how polymerization is controlled. Previously, we had determined that the stretch of unstructured residues N-terminal to the SAM of a Drosophila protein called polyhomeotic (Ph), a member of the polycomb group (PcG) of gene silencers, plays a key role in controlling Ph SAM polymerization. Ph SAM with its native linker created shorter polymers compared to Ph SAM attached to either a random linker or no linker. Here, we show that the SAM linker for the human Ph ortholog, polyhomeotic homolog 3 (PHC3), also controls PHC3 SAM polymerization but does so in the opposite fashion. PHC3 SAM with its native linker allows longer polymers to form compared to when attached to a random linker. Attaching the PHC3 SAM linker to Ph SAM also resulted in extending Ph SAM polymerization. Moreover, in the context of full-length Ph protein, replacing the SAM linker with PHC3 SAM linker, intended to create longer polymers, resulted in greater repressive ability for the chimera compared to wild-type Ph. These findings show that polymeric SAM linkers evolved to modulate a wide dynamic range of SAM polymerization abilities and suggest that rationally manipulating the function of SAM containing proteins through controlling their SAM polymerization may be possible.
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http://dx.doi.org/10.1021/bi3004318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045017PMC
July 2012

The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain.

J Biol Chem 2012 Mar 24;287(12):8702-13. Epub 2012 Jan 24.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3990, USA.

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.
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http://dx.doi.org/10.1074/jbc.M111.336115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308824PMC
March 2012

Identification of nucleic acid binding residues in the FCS domain of the polycomb group protein polyhomeotic.

Biochemistry 2011 Jun 12;50(22):4998-5007. Epub 2011 May 12.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, MSC 7760, San Antonio, Texas 78229-3990, United States.

Polycomb group (PcG) proteins maintain the silent state of developmentally important genes. Recent evidence indicates that noncoding RNAs also play an important role in targeting PcG proteins to chromatin and PcG-mediated chromatin organization, although the molecular basis for how PcG and RNA function in concert remains unclear. The Phe-Cys-Ser (FCS) domain, named for three consecutive residues conserved in this domain, is a 30-40-residue Zn(2+) binding motif found in a number of PcG proteins. The FCS domain has been shown to bind RNA in a non-sequence specific manner, but how it does so is not known. Here, we present the three-dimensional structure of the FCS domain from human Polyhomeotic homologue 1 (HPH1, also known as PHC1) determined using multidimensional nuclear magnetic resonance methods. Chemical shift perturbations upon addition of RNA and DNA resulted in the identification of Lys 816 as a potentially important residue required for nucleic acid binding. The role played by this residue in Polyhomeotic function was demonstrated in a transcription assay conducted in Drosophila S2 cells. Mutation of the Arg residue to Ala in the Drosophila Polyhomeotic (Ph) protein, which is equivalent to Lys 816 in HPH1, was unable to repress transcription of a reporter gene to the level of wild-type Ph. These results suggest that direct interaction between the Ph FCS domain and nucleic acids is required for Ph-mediated repression.
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http://dx.doi.org/10.1021/bi101487sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938326PMC
June 2011

Polycomb group targeting through different binding partners of RING1B C-terminal domain.

Structure 2010 Aug;18(8):966-75

Department of Biochemistry, University of Texas Health Science Center at San Antonio, MSC 7760, 7703 Floyd Curl Drive, San Antonio, TX 78229-3990, USA.

RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.
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http://dx.doi.org/10.1016/j.str.2010.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445678PMC
August 2010

Backbone resonance assignments of the 48 kDa dimeric putative 18S rRNA-methyltransferase Nep1 from Methanocaldococcus jannaschii.

Biomol NMR Assign 2009 Dec;3(2):251-4

Institut für Molekulare Biowissenschaften, Johann-Wolfgang-Goethe-Universität Frankfurt/M., Frankfurt, Germany.

Nep1 from Methanocaldococcus jannaschii is a 48 kDa dimeric protein belonging to the SPOUT-class of S-adenosylmethionine dependent RNA-methyltransferases and acting as a ribosome assembly factor. Mutations in the human homolog are the cause of Bowen-Conradi syndrome. We report here 1H, 15N and 13C chemical shift assignments for the backbone of the protein in its apo state.
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http://dx.doi.org/10.1007/s12104-009-9187-zDOI Listing
December 2009

The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site.

Nucleic Acids Res 2008 Mar 21;36(5):1542-54. Epub 2008 Jan 21.

Department of Biochemistry, X-ray Crystallography Core Laboratory, The University of Texas Health Science Center San Antonio, San Antonio, TX-78229, USA.

Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1(ts)) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 A resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 A resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 A resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase.
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http://dx.doi.org/10.1093/nar/gkm1172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275143PMC
March 2008

Prevention of metastases with a Mage-b DNA vaccine in a mouse breast tumor model: potential for breast cancer therapy.

Breast Cancer Res Treat 2005 May;91(1):19-28

Department of Cellular and Structural Biology, University of Texas Health Science Center, STCBM building, 15355 Lambda Drive, San Antonio, TX 78245, USA.

Anti-tumor vaccines are a relatively non-toxic alternative to conventional chemotherapeutic strategies to control breast cancer. Immunization with tumor-associated antigens (TAAs) triggers anti-tumor cytotoxic T lymphocytes (CTL), which can limit tumor progression. Here we report on the development and effectiveness of a TAA-based DNA vaccine encoding Mage-b1/2, the mouse homologue of the human MAGE-B1/2. As model system, we used immune competent Balb/c mice with syngeneic non-metastatic (64pT) or metastatic (4TO7cg) breast tumors. First, the presence of Mage-btranscripts in the 64pT and 4TO7cg breast tumors and metastases was demonstrated by RT-PCR, Southern blotting, and DNA sequencing. A DNA-based vaccine was developed from transcripts of one of the 64pT tumors, encoding the complete Mage-b1/2 protein, and subsequently tested for its preventive efficacy in both breast tumor models. Mice were immunized two times intramuscularly with the vaccine (pcDNA3.1-Mage-b1/2-V5), the control vector (pcDNA3.1-V5), or saline. Two weeks after the last immunization, the syngeneic 4TO7cg or 64pT tumor cell lines were injected in a mammary fat pad. Mice were monitored during the next 4 weeks for tumor formation, latency and size, and subsequently sacrificed for analysis. While the Mage-b1/2 vaccine had only a minor effect on the latency and growth of primary tumors, a significant and reproducible reduction in the number of 4TO7cg metastases was observed (vaccine versus control vector, p=0.0329; vaccine versus saline, p=0.0128). The observed protective efficacy of the Mage-b DNA vaccine correlated with high levels of vaccine-induced IFNgamma in spleen and lymph nodes upon re-stimulation in vitro. These results demonstrate the potential of TAA-based DNA vaccines in controlling metastatic disease in breast cancer patients.
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http://dx.doi.org/10.1007/s10549-004-6454-7DOI Listing
May 2005

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells.

Mol Cancer Ther 2004 Nov;3(11):1385-96

Department of Radiation Oncology, University of Texas Health Science Center, IDD Building, 14960 Omicron Drive, San Antonio, TX 78245, USA.

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.
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November 2004

Influence of radiotherapy on 6-sulphatoxymelatonin levels in the urine of brain cancer patients.

Neuro Endocrinol Lett 2000 ;21(3):203-207

Department of Radiation Oncology. The University of Texas Health Science Center, San Antonio, TX, USA.

OBJECTIVES: The synthesis of melatonin, an endogenous compound synthesized by the pineal gland in the brain, is reported to be depressed in patients with primary cancers of the breast, prostate, stomach and rectum. It is not known whether patients with brain cancer exhibit altered melatonin synthesis. Also unknown is whether radiotherapy given to the region of the brain where the pineal gland is located affects the synthesis of melatonin. This information could be relevant to the clinician for the successful treatment of brain cancer patients since melatonin has been reported to be a potent oncostatic agent. METHODS: Urinary levels of 6-sulphatoxymelatonin, the chief metabolite of melatonin, are routinely used as an index of pineal melatonin production and secretion. In this study, the concentrations of 6-sulphatoxymelatonin (aMT6S) excreted in the urine before and during radiotherapy of patients with primary or metastatic brain cancer were determined and compared with the values obtained in breast or lung cancer patients who also received radiotherapy (excluding exposure of the brain where the pineal gland is located). RESULTS: The results showed a wide variation in the mean concentration of aMT6S excreted in the urine. CONCLUSION: The data from this preliminary study suggested that radiotherapy given to the region of human brain, where the pineal gland is located, does not significantly affect the excretion of aMT6S, the chief metabolite of melatonin.
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January 2000