Biomedical subjects

Sangpen Chamnongpol

Publications and source records attributed to Sangpen Chamnongpol.

6 recordsLinked to original sources

PubMed2006

Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways.

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

Amino Acid Sequence↗

PubMed2005

Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves.

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H(2)O(2) and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.

Catalase↗

PubMed2004

SH3 domain protein-binding arrays.

First identified as part of the Rous sarcoma oncogene product Src, SH3 (Src Homology 3) domains play an important role in intercellular communication and intracellular signal transduction. A high-throughput assay for ligand binding to SH3 domains--SH3 domain proteins immobilized on a membrane--allows rapid visualization of numerous SH3 domain protein-protein interactions with no expensive equipment or radioactivity required. Once the array is constructed or obtained commercially, the procedure is straightforward: The protein of interest is cloned into a fusion-tagged expression vector and expressed in bacteria, the prepared bacterial extract is incubated with the array membrane, and the signal is measured using a chemiluminescence detection system.

Humans↗

PubMed2003

Mg2+ sensing by the Mg2+ sensor PhoQ of Salmonella enterica.

The PhoP/PhoQ two-component regulatory system governs the adaptation to low Mg(2+) environments and virulence in several Gram-negative species. During growth in low Mg(2+), the sensor PhoQ modifies the activity of the response regulator PhoP promoting gene transcription, whereas growth in high Mg(2+) represses transcription of PhoP-activated genes. The PhoQ protein harbors a periplasmic domain of 146 amino acid residues that binds Mg(2+) in vitro and is required for Mg(2+)-mediated repression in vivo. Here, we identify periplasmic mutants of the Salmonella PhoQ protein that allow transcription of PhoP-activated genes even under high Mg(2+) concentrations. When expressed in a strain harboring a PhoP variant that is phosphorylated from acetyl phosphate, some of the mutants failed to repress PhoP-promoted transcription in high Mg(2+), whereas others displayed a wild-type ability to do so. Mutant PhoQ proteins that allowed expression of PhoP-activated genes in high Mg(2+) displayed a pattern of iron-mediated cleavage in vitro that was different from that displayed by wild-type PhoQ, indicative of altered Mg(2+) binding. A PhoQ protein with the conserved histidine residue (H277) substituted by alanine could not promote transcription of PhoP-activated genes in low Mg(2+) but could turn off expression in response to high Mg(2+). Our studies demonstrate that residues G93, W97, H120 and T156 are required for a wild-type response to Mg(2+), and suggest that Mg(2+) binding to the periplasmic domain regulates several activities in the PhoQ protein.

Amino Acid Sequence↗

PubMed2002

Mg2+ homeostasis and avoidance of metal toxicity.

Because Mg2+ is required for a wide variety of cellular functions, its intracellular levels must be tightly regulated. In the Gram-negative bacterium Salmonella enterica, three transporters mediate Mg2+ uptake: the P-type ATPases MgtA and MgtB, whose expression is transcriptionally induced in low Mg2+ by the Mg2+-regulated PhoP/PhoQ two-component system; and CorA, whose transcription is regulated neither by the levels of Mg2+ nor by the PhoP/PhoQ system. We now report that mutants defective in phoP or in both mgtA and mgtB are hypersensitive to oxidative stress-dependent Fe (II)-mediated killing. These mutants display increased iron accumulation and heightened Ni2+ uptake. Inactivation of the corA gene restored Fe(II) resistance to the phoP mutant and eliminated uptake of Ni2+. Neither corA transcription nor CorA protein levels were altered in the phoP mutant, suggesting that CorA alters its activity in response to the presence of PhoP-regulated determinants. Downregulation of CorA activity in low Mg2+ environments may enable Salmonella to avoid the uncontrolled influx of toxic metals.

Ascorbic Acid↗

PubMed2002

Fe(III)-mediated cellular toxicity.

Because it can undergo reversible changes in oxidation state, iron is an excellent biocatalyst but also a potentially deleterious metal. Iron-mediated toxicity has been ascribed to Fe(II), which reacts with oxygen to generate free radicals that damage macromolecules and cause cell death. However, we now report that Fe(III) exhibits microbicidal activity towards strains of Salmonella enterica, Escherichia coli and Klebsiella pneumoniae defective in the Fe(III)-responding PmrA/PmrB signal transduction system. Fe(III) bound to a pmrA Salmonella mutant more effectively than to the isogenic wild-type strain and exerted its microbicidal activity even under anaerobic conditions. Moreover, Fe(III) permeabilized the outer membrane of the pmrA mutant, rendering it susceptible to vancomycin, which is normally non-toxic to Gram-negative species. On the other hand, Fe(III) did not affect the viability of a mutant defective in Fur, the major regulator of cytosolic iron homeostasis, which is hypersensitive to Fe(II)-mediated toxicity. A functional pmrA gene was necessary for bacterial survival in soil. Our results indicate that Fe(III) exerts its microbicidal activity by a mechanism that is oxygen independent and different from that mediated by Fe(II).

Bacterial Proteins↗