Biomedical subjects

Francesc R Garcia-Gonzalo

Publications and source records attributed to Francesc R Garcia-Gonzalo.

5 recordsLinked to original sources

PubMed2006

TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2.

Amino Acid Sequence↗

PubMed2006

Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel.

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.

Acrylamides↗

PubMed2005

Requirement of phosphatidylinositol-4,5-bisphosphate for HERC1-mediated guanine nucleotide release from ARF proteins.

HERC1 is a giant multidomain protein involved in membrane trafficking through its interaction with vesicle coat proteins such as clathrin and ARF. Previously, it has been shown that the RCC1-like domain 1 (RLD1) of HERC1 stimulates guanine nucleotide dissociation on ARF1 and Rab proteins. In this study, we have analyzed whether HERC1 may also regulate ARF6 activity. We show that HERC1, through its RLD1, stimulates GDP release from ARF6 but, unexpectedly, it inhibits GDP/GTP exchange on ARF6 under conditions where ARNO stimulates it. Furthermore, we demonstrate that the activity of HERC1 as a guanine nucleotide release factor requires the presence of PI(4,5)P(2) bound to HERC1's RLD1. In agreement with this, we find that purified HERC1 contains PI(4,5)P(2) bound to the RLD1.

ADP-Ribosylation Factor 6↗

PubMed2004

The giant protein HERC1 is recruited to aluminum fluoride-induced actin-rich surface protrusions in HeLa cells.

HERC1 is a very large protein involved in membrane traffic through both its ability to bind clathrin and its guanine nucleotide exchange factor (GEF) activity over ARF and Rab family GTPases. Herein, we show that HERC1 is recruited onto actin-rich surface protrusions in ARF6-transfected HeLa cells upon aluminum fluoride (AlF(4)(-)) treatment. Moreover, the fact that HERC1 overexpression does not stimulate protrusion formation in the absence of AlF(4)(-), in conditions where ARNO does, indicates that HERC1 is not acting as an ARF6-GEF in this system, but that instead its recruitment takes place downstream of ARF6 activation. Finally, we suggest a phosphoinositide-binding mechanism whereby HERC1 may translocate to these protrusions.

ADP-Ribosylation Factor 6↗

PubMed2003

Interaction between HERC1 and M2-type pyruvate kinase.

HERC proteins are characterized by having one or more RCC1-like domains as well as a C-terminal HECT domain in their amino acid sequences. This has led researchers to suggest that they may act as both guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we describe a physical interaction between the HECT domain of HERC1, a giant protein involved in intracellular membrane traffic, and the M2 isoform of glycolytic enzyme pyruvate kinase (M2-PK). Partial colocalization of endogenous proteins was observed by immunofluorescence studies. This interaction neither induced M2-PK ubiquitination nor affected its enzymatic activity. The putative significance of the association is discussed.

Adaptor Proteins, Signal Transducing↗