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Luis S Mayorga

Publications and source records attributed to Luis S Mayorga.

11 recordsLinked to original sources
PubMed2006

Development of a Premature Stop Codon-detection method based on a bacterial two-hybrid system.

BACKGROUND: The detection of Premature Stop Codons (PSCs) in human genes is very useful for the genetic diagnosis of different hereditary cancers, e.g. Familial Breast Cancer and Hereditary Non-Polyposis Colorectal Cancer (HNPCC). The products of these PSCs are truncated proteins, detectable in vitro by the Protein Truncation Test and in vivo by using the living translation machinery of yeast or bacteria. These living strategies are based on the construction of recombinant plasmids where the human sequence of interest is inserted upstream of a reporter gene. Although simple, these assays have their limitations. The yeast system requires extensive work to enhance its specificity, and the bacterial systems yield many false results due to translation re-initiation events occurring post PSCs. Our aim was to design a recombinant plasmid useful for detecting PSCs in human genes and resistant to bacterial translation re-initiation interferences. RESULTS: A functional recombinant plasmid (pREAL) was designed based on a bacterial two-hybrid system. In our design, the in vivo translation of fused fragments of the Bordetella pertussis adenylate cyclase triggers the production of cAMP giving rise to a selectable bacterial phenotype. When a gene of interest is inserted between the two fragments, any PSC inhibits the enzymatic activity of the product, and translation re-initiation events post-PSC yield separated inactive fragments. We demonstrated that the system can accurately detect PSCs in human genes by inserting mutated fragments of the brca1 and msh2 gene. Western Blot assays revealed translation re-initiation events in all the tested colonies, implying that a simpler plasmid would not be resistant to this source of false negative results. The application of the system to a HNPCC family with a nonsense mutation in the msh2 gene correctly diagnosed wild type homozygous and heterozygous patients. CONCLUSION: The developed pREAL is applicable to the detection of PSCs in human genes related to different diseases and is resistant to translation re-initiation events. The diagnosis steps are easy, have a low cost, detect only pathologic mutations, and allow the analysis of separated alleles.

Codon, Nonsense↗
PubMed2006

Calcium-induced acrosomal exocytosis requires cAMP acting through a protein kinase A-independent, Epac-mediated pathway.

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.

Acrosome Reaction↗
PubMed2006

Rab22a regulates the sorting of transferrin to recycling endosomes.

Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16 degrees C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes.

Animals↗
PubMed2005

Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis.

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCbetaII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain--a key region for the function of synaptotagmins--are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.

Acrosome↗
PubMed2005

Dynamics of SNARE assembly and disassembly during sperm acrosomal exocytosis.

The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle--the acrosome--is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/alpha-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/alpha-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)-specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.

Acrosome↗
PubMed2005

[Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis].

MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of penetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tyrosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tyrosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomatically the family members, avoiding sending all the samples to foreign centers.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus.

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).

Animals↗
PubMed2002

The intraacrosomal calcium pool plays a direct role in acrosomal exocytosis.

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP(3)-sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP(3)-sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.

Acrosome↗
PubMed2002

A PCR-mutagenesis strategy for rapid detection of mutations in codon 634 of the ret proto-oncogene related to MEN 2A.

BACKGROUND: Multiple endocrine neoplasias type 2A (MEN 2A) is a dominantly inherited cancer syndrome. Missence mutations in the codon encoding cysteine 634 of the ret proto-oncogene have been found in 85% of the MEN 2A families. The main tumour type always present in MEN 2A is medullar thyroid carcinoma (MTC). Only 25% of all MTC are hereditary, and generally they are identified by a careful family history. However, some familial MTCs are not easily detected by this means and underdiagnosis of MEN 2A is suspected. METHODS: DNA samples from MEN 2A patients were amplified by PCR. The products were incubated with the restriction enzyme Bst ApI or Bgl I. The samples were loaded in non-denaturing 10% Polyacrilamyde Gel and run at 120 volts for 40 min. The gels were stained with 10 microg/ml ethidium bromide, and the bands were visualized under a UV lamp. RESULTS: We developed a PCR-mutagenic method to check the integrity of the three bases of the cysteine 634 codon. CONCLUSION: The method can be used to detect inherited mutations in MTC patients without a clear family history. The method is relatively simple to use as a routine test in these patients to decrease the underdiagnosis of MEN 2A. In addition, the assay can be used to screen affected families with any mutation in cysteine 634.

Journal Article↗
PubMed2002

SNARE complex assembly is required for human sperm acrosome reaction.

Exocytosis of the acrosome (the acrosome reaction) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg. Ca(2+) is an essential mediator of this regulated secretory event. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, NSF, and synaptotagmin VI in the human sperm acrosome reaction. Interestingly, Rab3A elicits an exocytotic response of comparable magnitude to that of Ca(2+). Here, we report a direct role for the SNARE complex in the acrosome reaction. First, the presence of SNARE proteins is demonstrated by Western blot. Second, the Ca(2+)-triggered acrosome reaction is inhibited by botulinum neurotoxins BoNT/A, -E, -C, and -F. Third, antibody inhibition studies show a requirement for SNAP-25, SNAP-23, syntaxins 1A, 1B, 4, and 6, and VAMP 2. Fourth, addition of bacterially expressed SNAP-25 and SNAP-23 abolishes exocytosis. Acrosome reaction elicited by Rab3-GTP is also inhibited by BoNT/A, -C, and -F. Taken together, these results demonstrate a requirement for members of all SNARE protein families in the Ca(2+)- and Rab3A-triggered acrosome reaction. Furthermore, they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes.

Acrosome Reaction↗