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Biomedical subjects

Philip J Thomas

Publications and source records attributed to Philip J Thomas.

At least 19 recordsLinked to original sources
PubMed2006

Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3- secretion: relevance to cystic fibrosis.

Fluid and HCO(3)(-) secretion are vital functions of the pancreatic duct and other secretory epithelia. CFTR and Cl(-)/HCO(3)(-) exchange activity at the luminal membrane are required for these functions. The molecular identity of the Cl(-)/HCO(3)(-) exchangers and their relationship with CFTR in determining fluid and HCO(3)(-) secretion are not known. We show here that the Cl(-)/HCO(3)(-) exchanger slc26a6 controls CFTR activity and ductal fluid and HCO(3)(-) secretion. Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO(3)(-) secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTR(inh)-172, knock-down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6(-/-) duct cells revealed that deletion of slc26a6 resulted in dis-regulation of CFTR activity by removal of tonic inhibition of CFTR by slc26a6. These findings reveal the intricate regulation of CFTR activity by slc26a6 in both the resting and stimulated states and the essential role of slc26a6 in pancreatic HCO(3)(-) secretion in vivo.

Animals↗
PubMed2006

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome.

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.

Adenosine Triphosphate↗
PubMed2006

Doxazosin and serotonin (5-HT) receptor (1A, 2A, and 4) antagonists inhibit 5-HT-mediated human cavernosal contraction.

Penile erection results from the balance between relaxation and contractile mechanisms of the corpus cavernosum. Only a few studies suggest a role for endogenous contractile agents such as 5-hydroxytryptamine (5-HT). Our aim was to confirm the possible role of 5-HT in human erection. The effect of 5-HT on human cavernosal tissues, as well as those of doxazosin (shown previously to have 5-HT inhibitory action), ketanserin (5-HT (2A) receptor antagonist), NAN-190 (5-HT (1A) receptor antagonist), and SB 203186 (5-HT (4) receptor antagonist) on 5-HT-mediated effects, were assessed using the organ bath technique, including electrical field stimulation study (EFS). Results are presented as median (mg/mg = mg contraction/mg of tissue). Consistent 5-HT-mediated (10(-3) M) contractions were demonstrated (n = 18; 63 mg/mg). These contractions were inhibited with ketanserin by 90% (n = 8), NAN-190 by 68% (n = 12), and SB 203186 by 55% (n = 12). Doxazosin showed a similar 5-HT inhibitory action in a concentration-dependent manner (10(-4) M; 94% reduction; n = 8, 10(-6) M; 68.3% reduction; n = 8). Our EFS studies indicated the presence of neuronally derived 5-HT and that a majority of the nonnoradrenogenic contraction (54%) was mediated via 5-HT(2A) receptors. These findings suggest that 5-HT may play a role in the human detumescence process via 5-HT(1A), 5-HT(2A), and 5-HT(4) receptors. Neuronally released 5-HT is probably an important contractile neurotransmitter in the erectile process. Doxazosin, ketanserin, and 5-HT(1A) and 5-HT(4) receptor antagonists may be useful as part of combination therapy used to treat erectile dysfunction.

Adult↗
PubMed2006

Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6.

The SLC26 transporters are a family of mostly luminal Cl- and HCO3- transporters. The transport mechanism and the Cl-/HCO3- stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3- and Cl- fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl-/1HCO3- exchanger. The membrane potential modulated the apparent affinity for extracellular Cl- of Cl-/HCO3- exchange by slc26a3. Interestingly, the replacement of Cl- with NO3- or SCN- uncoupled the transport, with large NO3- and SCN- currents and low HCO3- transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3--buffered Cl--free media. These findings were used to develop a turnover cycle for Cl- and HCO3- transport by slc26a3. Cl- and HCO3- flux measurements revealed that slc26a6 mediates a 1Cl-/2HCO3- exchange. Accordingly, holding the membrane potential at 40 and -100 mV accelerated and inhibited, respectively, Cl--mediated HCO3- influx, and holding the membrane potential at -100 mV increased HCO3--mediated Cl- influx. These findings indicate that slc26a6 functions as a coupled 1Cl-/2HCO3- exchanger. The significance of isoform-specific Cl- and HCO3- transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl- absorption and HCO3- secretion.

Animals↗
PubMed2006

Regulatory interaction between CFTR and the SLC26 transporters.

Most epithelia that express CFTR secrete fluid rich in HCO3- and poor in Cl- that is generated by a CFTR-dependent Cl- absorption and HCO3- secretion process that when aberrant leads to human diseases such as cystic fibrosis and congenital chloride diarrhoea. Epithelial Cl- absorption and HCO3- secretion require expression of CFTR and other Cl- and HCO3- transporters in the luminal membrane of the secreting cells. Recent advances in understanding this critical epithelial function revealed that the luminal Cl- and HCO3- transporters are members of the SLC26 family. Characterization of several members of the family reveals that all characterized thus far are electrogenic with an isoform specific Cl-/HCO3- transport stoichiometry. In vivo these transporters exist in a transporting complex with CFTR. The SLC26 transporters and CFTR are recruited to the complex by binding to scaffolds containing PDZ domains. Upon stimulation and PKA-dependent phosphorylation of CFTR R domain, the R domain binds to the SLC26 transporter STAS domain. Interaction of the R and STAS domains results in a marked and mutual activation of CFTR and the SLC26 transporters. The significance of this mode of regulation to epithelial Cl- absorption and HCO3- secretion is obvious.

Animals↗
PubMed2005

A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.

Parkinson disease and other alpha-synucleinopathies are characterized by the deposition of intraneuronal alpha-synuclein (alphaSyn) inclusions. A significant fraction (about 15%) of alphaSyn in these pathological structures are truncated forms that have a much higher propensity than the full-length alphaSyn to form aggregates in vitro. However, little is known about the role of truncated alphaSyn species in pathogenesis or the means by which they are generated. Here, we have provided an in vitro mechanistic study demonstrating that truncated alphaSyns induce rapid aggregation of full-length protein at substoichiometric ratios. Co-overexpression of truncated alphaSyn with full-length protein increases cell vulnerability to oxidative stress in dopaminergic SH-SY5Y cells. These results suggest a precipitating role for truncated alphaSyn in the pathogenesis of diseases involving alphaSyn aggregation. In this regard, the A53T mutation found in some cases of familial Parkinson disease exacerbates the accumulation of insoluble alphaSyns that correlates with the onset of pathology in transgenic mice expressing human alphaSyn-A53T mutant. The caspase-like activity of the 20 S proteasome produces truncated fragments similar to those found in patients and animal models from degradation of unstructured alphaSyn. We propose a model in which incomplete degradation of alphaSyn, especially under overloaded proteasome capacity, produces highly amyloidogenic fragments that rapidly induce the aggregation of full-length protein. These aggregates in turn reduce proteasome activity, leading to further accumulation of fragmented and full-length alphaSyns, creating a vicious cycle of cytotoxicity. This model has parallels in other neurodegenerative diseases, such as Huntington disease, where coaggregation of poly(Q) fragments with full-length protein has been observed.

Aged↗
PubMed2005

Nucleotide binding domain interactions during the mechanochemical reaction cycle of ATP-binding cassette transporters.

ATP-binding cassette (ABC) transporters serve as importers and exporters for a wide variety of solutes in both prokaryotes and eukaryotes, and are implicated in microbial drug resistance and a number of significant human genetic disorders. Initial crystal structures of the soluble nucleotide binding domains (NBDs) of ABC transporters, while a significant step towards understanding the coupling of ATP binding and hydrolysis to transport, presented researchers with important questions surrounding the role of the signature sequence residues, the composition of the nucleotide binding sites, and the mode of NBD dimerization during the transport reaction cycle. Recent studies have begun to address these concerns. This mini-review summarizes the biochemical and structural characterizations of two archaebacterial NBDs from Methanocaldococcus jannaschii, MJ0796 and MJ1267, and offers current perspectives on the functional mechanism of ABC transporters.

ATP-Binding Cassette Transporters↗
PubMed2005

Recognition and processing of misfolded proteins by PA700, the 19S regulatory complex of the 26S proteasome.

The 26S proteasome is composed of the core 20S proteasome in association with the 19S regulatory complex, or PA700. PA700 has multiple activities, including ATPase activity, polyubiquitin-chain binding activity, deubiquitination activity, chaperone-like activity, and substrate remodeling activity. The concerted action of these activities leads to efficient degradation of protein substrates by the 26S proteasome. In this chapter we describe protocols for purifying PA700 and the 20S complexes from bovine red cells and present methods to assay the chaperone-like activity and the substrate remodeling activity of PA700.

Animals↗
PubMed2005

Aggresome formation.

Bulk protein degradation in the cell is catalyzed by the ubiquitin-proteasome system (UPS). At the heart of the UPS is the proteasome, a large multisubunit tightly-regulated protease. The UPS performs key functions in protein quality control by monitoring and eliminating potentially toxic misfolded or damaged proteins. When the capacity of this protease system is exceeded, misfolded protein substrates aggregate and are assembled through an active and regulated process to form an aggresome. Aggresomes are dynamic structures, formed as a general response to an overload of improperly folded proteins. Assembly of aggresomes occurs at the centrosome, a perinuclear structure that also serves as a site for the recruitment and concentration of components of the UPS, including the proteasome, its regulators, and other proteins typically involved in protein quality control. Thus, in addition to other cellular activities, the centrosome may play a central role in protein quality control, sitting at the crossroads of protein folding, degradation, and aggregation.

Cell Line↗
PubMed2004

Side chain and backbone contributions of Phe508 to CFTR folding.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

Animals↗
PubMed2004

Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein.

Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.

Animals↗
PubMed2004

Gating of CFTR by the STAS domain of SLC26 transporters.

Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NP(o)) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.

Antiporters↗
PubMed2004

Dynamic control of cystic fibrosis transmembrane conductance regulator Cl(-)/HCO3(-) selectivity by external Cl(-).

HCO(3)(-) secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pH(i) and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR Cl(-)/HCO(3)(-) permeability ratio, which is regulated by external Cl(-) (Cl(-)(o)). Thus, reducing external Cl(-) from 110 to 0-10 mm resulted in the expected increase in membrane potential, but with no corresponding OH(-) or HCO(3)(-) influx. Approximately 3-4 min after reducing Cl(o)(-) to 0 mm, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH(-) and HCO(3)(-) influx. The switch in membrane permeability to OH(-)/HCO(3)(-) can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH(-) influx in response to elevating pH(o) to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at Cl(o)(-) of 5 mm. Conversely, re-addition of external Cl(-) after the switch in membrane potential did not stop HCO(3)(-) influx, which continued for about 3.9 min after Cl(-) addition. Importantly, addition of external Cl(-) to cells incubated in Cl(-)-free medium never resulted in HCO(3)(-) efflux. Voltage and current clamp experiments showed that the delayed HCO(3)(-) transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl(-) only and a Cl(-) and OH(-)/HCO(3)(-) permeable state. The switch between the states is controlled by external Cl(-). Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl(-)-containing and Cl(-)-free media. The physiological significance of these finding is discussed in the context of HCO(3)(-) secretion by tissues such as the pancreas and salivary glands.

Animals↗
PubMed2003

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Adenosine Diphosphate↗
PubMed2003

Organic solutes rescue the functional defect in delta F508 cystic fibrosis transmembrane conductance regulator.

The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (Delta F508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of Delta F508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of Delta F508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested Delta F508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing Delta F508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-Delta F508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-Delta F508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the Delta F508 and wild type CFTR and inhibited Delta F508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36Cl- efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of Delta F508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of Delta F508 CFTR.

Animals↗
PubMed2002

Endoproteolytic activity of the proteasome.

The proteasome plays a central role in the degradation of regulatory and misfolded proteins. Current models suggest that substrates access the internal catalytic sites by processively threading their termini through the gated substrate channel. Here, we found that latent (closed) and activated (open) proteasomes degraded two natively disordered substrates at internal peptide bonds even when they lacked accessible termini, suggesting that these substrates themselves promoted gating of the proteasome. This endoproteolysis provides a molecular mechanism for regulated release of transcription factors from inactive precursors as well as a means of accessing internal folding defects of misfolded multidomain proteins.

Amino Acid Sequence↗
PubMed2002

A molecular mechanism for aberrant CFTR-dependent HCO(3)(-) transport in cystic fibrosis.

Aberrant HCO(3)(-) transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl(-)-dependent HCO(3)(-) transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO(3)(-) current by CFTR cannot account for CFTR-activated HCO(3)(-) transport and that CFTR does not activate AE1-AE4. In contrast, CFTR markedly activates Cl(-) and OH(-)/HCO(3)(-) transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform-specific stoichiometries. DRA activity occurred at a Cl(-)/HCO(3)(-) ratio > or =2. SLC26A6 activity is voltage regulated and occurred at HCO(3)(-)/Cl(-) > or =2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO(3)(-) transport (one SLC26 transporter-electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain-electroneutral transport), the CF-associated aberrant HCO(3)(-) transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.

Amino Acid Sequence↗