Phenotypic switch of smooth muscle cells in paediatric chronic intestinal pseudo-obstruction syndrome.

Smooth Muscle Cells (SMC) are unique amongst all muscle cells in their capacity to modulate their phenotype. Indeed, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate, switching between a quiescent contractile state and a highly proliferative and migratory phenotype, a quality often associated to SMC dysfunction. However, phenotypic plasticity remains poorly examined in the field of gastroenterology in particular in pathologies in which gut motor activity is impaired. Here, we assessed SMC status in biopsies of infants with chronic intestinal pseudo-obstruction (CIPO) syndrome, a life-threatening intestinal motility disorder. We showed that CIPO-SMCs harbour a decreased level of contractile markers. This phenotype is accompanied by an increase in Platelet-Derived Growth Factor Receptor-alpha (PDGFRA) expression. We showed that this modulation occurs without origin-related differences in CIPO circular and longitudinal-derived SMCs. As we characterized PDGFRA as a marker of digestive mesenchymal progenitors during embryogenesis, our results suggest a phenotypic switch of the CIPO-SMC towards an undifferentiated stage. The development of CIPO-SMC culture and the characterization of SMC phenotypic switch should enable us to design therapeutic approaches to promote SMC differentiation in CIPO.

Epithelial Splicing Regulatory Protein 1 (ESRP1) is a new regulator of stomach smooth muscle development and plasticity.

In vertebrates, stomach smooth muscle development is a complex process that involves the tight transcriptional or post-transcriptional regulation of different signalling pathways. Here, we identified the RNA-binding protein Epithelial Splicing Regulatory Protein 1 (ESRP1) as an early marker of developing and undifferentiated stomach mesenchyme. Using a gain-of-function approach, we found that in chicken embryos, sustained expression of ESRP1 impairs stomach smooth muscle cell (SMC) differentiation and FGFR2 splicing profile. ESRP1 overexpression in primary differentiated stomach SMCs induced their dedifferentiation, promoted specific-FGFR2b splicing and decreased FGFR2c-dependent activity. Moreover, co-expression of ESRP1 and RBPMS2, another RNA-binding protein that regulates SMC plasticity and Bone Morphogenetic Protein (BMP) pathway inhibition, synergistically promoted SMC dedifferentiation. Finally, we also demonstrated that ESRP1 interacts with RBPMS2 and that RBPMS2-mediated SMC dedifferentiation requires ESRP1. Altogether, these results show that ESRP1 is expressed also in undifferentiated stomach mesenchyme and demonstrate its role in SMC development and plasticity.

Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration.

Temperature and pressure effects on C112S azurin: volume, expansivity, and flexibility changes.

The pressure-induced unfolding of the mutant C112S azurin from Pseudomonas aeruginosa was monitored both under steady state and dynamic conditions. The unfolding profiles were obtained by recording the spectral shift of the fluorescence emission as well as by phosphorescence intensity measurements. We evaluated the difference in free energy, ΔG, as a function of pressure and temperature. The dependence of ΔG on temperature showed concave profile at all pressures studied. A positive heat capacity change of about 4.3 kJ mol(-1) deg(-1) fitted all the curves. The volume change of the reaction showed a moderate dependence on temperature when compared with other proteins previously studied. The kinetic activation parameters (ΔV*, ΔH*, ΔS*) were obtained from upward and downward pressure-jump experiments and used to characterize the volumetric and energetic properties of the transition state between native and unfolded protein. Our findings suggest that the folding and unfolding reaction paths passed through different transition states. The change in the phosphorescence lifetime with pressure pointed out that pressure-induced unfolding occurred within two steps: the first leading to an increased protein flexibility, presumably caused by water penetration into the protein. Major structural changes of the tryptophan environment occurred in a second step at higher pressures.

Biotherapeutic approaches against cardio-metabolic dysfunctions based on extracellular vesicles.

Among the different pathways involved in the cell-to-cell communication, extracellular vesicles (EVs) are defined as key players in the transport of different signalling molecules, such as lipids, proteins, and RNA, from the originating cells to specific target cells. The biogenesis and composition of EVs are complex and confer them a unique ability to more effectively reach tissues and cells as compared to other types of synthetic carriers. Owing to these properties, EVs have been suggested as new therapeutic tools for personalized medicine. Since cardiometabolic diseases have reached pandemic proportions, new therapies are needed to be developed. In this context, EVs appear as promising therapeutic tools against cardiometabolic disorders associated with obesity and diabetes. This review focuses on the latest research on preclinical applications of EVs for cardiometabolic diseases, and draw primarily on our experience in this area.

Regenerating islet-derived 1α (Reg-1α) protein is new neuronal secreted factor that stimulates neurite outgrowth via exostosin Tumor-like 3 (EXTL3) receptor.

Regenerating islet-derived 1α (Reg-1α)/lithostathine, a member of a family of secreted proteins containing a C-type lectin domain, is expressed in various organs and plays a role in proliferation, differentiation, inflammation, and carcinogenesis of cells of the digestive system. We previously reported that Reg-1α is overexpressed during the very early stages of Alzheimer disease, and Reg-1α deposits were detected in the brain of patients with Alzheimer disease. However, the physiological function of Reg-1α in neural cells remains unknown. Here, we show that Reg-1α is expressed in neuronal cell lines (PC12 and Neuro-2a) and in rat primary hippocampal neurons (E17.5). Reg-1α is mainly localized around the nucleus and at the membrane of cell bodies and neurites. Transient overexpression of Reg-1α or addition of recombinant Reg-1α significantly increases the number of cells with longer neurites by stimulating neurite outgrowth. These effects are abolished upon down-regulation of Reg-1α by siRNA and following inhibition of secreted Reg-1α by antibodies. Moreover, Reg-1α colocalizes with exostosin tumor-like 3 (EXTL3), its putative receptor, at the membrane of these cells. Overexpression of EXTL3 increases the effect of recombinant Reg-1α on neurite outgrowth, and Reg-1α is not effective when EXTL3 overexpression is down-regulated by shRNA. Our findings indicate that Reg-1α regulates neurite outgrowth and suggest that this effect is mediated by its receptor EXTL3.

Effects of pressure and osmolytes on the allosteric equilibria of Salmonella typhimurium tryptophan synthase.

Osmolytes are common constituents of bacteria that may be produced or accumulate at high concentrations, up to 1 M, when cells are subjected to stresses like ionic strength and temperature. However, the effects of osmolytes on the allosteric properties of bacterial enzymes have rarely been examined. We have studied the effects of osmolytes and hydrostatic pressure on the allosteric equilibria of Salmonella typhimurium tryptophan (Trp) synthase. Trp synthase is a well-studied multienzyme complex with activity tightly regulated by allosteric interactions between the α- and ß-subunits. Trp synthase activity is affected by a wide range of physical parameters, including monovalent cations, pH, ligands, solvents, and hydrostatic pressure. Osmolytes, including betaine, taurine, sucrose, and polyethylene glycol, activate Trp synthase 2-3-fold in the absence of monovalent cations, indicating that osmolytes can stabilize the active closed conformation. However, in the presence of monovalent cations, osmolytes have only minor effects on activity and allosteric equilibria, but 1 M betaine stabilizes the Trp synthase-Ser-indoline complex against apparent pressure-induced subunit dissociation. Na(+) and K(+) are more effective at shifting the α-aminoacrylate-indoline quinonoid equilibrium toward the quinonoid side, with a K(Q) of 8-10, than NH(4)(+)(K(Q) ~ 2). Furthermore, pressure-jump experiments show that the mechanism of indoline reaction to form a quinonoid complex may be different for the NH(4)(+) enzyme than the Na(+) and K(+) forms. These results show that osmolytes have subtle but significant effects on the allosteric properties of Trp synthase, and these effects may be important in vivo.

Time-course and regional analyses of the physiopathological changes induced after cerebral injection of an amyloid ß fragment in rats.

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid ß protein (Aß) formed by pathological processing of the Aß precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aß fragment 25-35 (Aß(25-35)) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aß(25-35) peptide was used as negative control. The aggregated forms of Aß peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aß(25-35) decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aß(25-35), the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aß(25-35) injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD.

Staphylococcal enterotoxin A: Partial unfolding caused by high pressure or denaturing agents enhances superantigenicity.

The effect of transient exposure of Staphylococcus aureus enterotoxin A (SEA) to high pressure and/or denaturing agents was examined by assessing the toxin superantigenicity and immunoreactivity, and by monitoring pressure-induced changes in fluorescence emission spectra. Pressurization of SEA at 600 MPa and 45 degrees C in Tris-HCl buffer (20 mM, pH 7.4) resulted in a marked increase in both T-cell proliferation (superantigenicity) and immunoreactivity. In opposite, pressurization at 20 degrees C did not change significantly SEA superantigenicity and immunoreactivity, indicating some toxin baro-resistance. Exposure of SEA to 8 M urea at atmospheric pressure or at 600 MPa and 20 degrees C, also led to a marked increase of superantigenicity (but not of immunoreactivity). In contrast, exposure of SEA to sodium-dodecylsulfate (30 mM) led to an increase of immunoreactivity with some effect on superantigenicity after pressurization at 45 degrees C only. High pressure up to 600 MPa induced spectral changes which at 20 degrees C were fully reversible upon decompression. At 45 degrees C, however, a sharp break of the centre of spectral mass mainly due to tryptophan residues was observed at 300 MPa, and irreversible spectral changes mainly related to tyrosine residues subsisted after pressure release, indicating a marked protein conformational transition. Urea 8 M further increased SEA structural changes at 600 MPa and 20 degrees C. These results indicate that SEA, under a combination of high pressure and mild temperature, as well as in the presence of urea, partly unfolds to a structure of strongly increased T-cell proliferative ability.

Structure-function perturbation and dissociation of tetrameric urate oxidase by high hydrostatic pressure.

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.

Asymmetric kinetics of protein structural changes.

Thermodynamic and kinetic understanding of structural transformations in proteins is critical to new developments in medicine and biotechnology. These fields often require the design of mechanism-based modulators of protein function. Researchers increasingly consider these structural changes-such as folding/unfolding or shuttling between active and inactive states-within the energy landscape concept that supposes a high-dimensional, rugged conformational surface. The unevenness, or asperity, of this conformational surface results from energetic barriers and kinetic traps. However, for a large number of protein reactions, such as reversible folding/unfolding, the literature only reports simple two-state transitions, which calls into question the use of a more complex energy landscape model. The question is: are these reactions really that simple, or are we misled by a biased experimental approach? In this Account, we argue in favor of the latter possibility. Indeed, the frequently employed temperature-jump method only allows recording protein structure changes in the heating direction. Under those conditions, it might not be possible to detect other kinetic pathways that could have been taken in the cooling direction. Recently, however, we have developed bidirectional pressure- and temperature-jump methods, which can offer new insights. Here, we show the potential of these methods both for studying protein folding/unfolding reactions, taking ribonuclease A as model, and for studying functionally relevant protein conformational changes, using the open/closed allosteric transition of tryptophan synthase. For example, the heating and cooling temperature-jump induced kinetics involved in the folding/unfolding conformational surface of ribonuclease A is illustrated above. In both of our model systems, the kinetic transition states of several reaction steps were path-dependent, i.e. the rates and thermodynamic activation parameters depend on the direction of the applied pressure and temperature perturbation. This asymmetry suggests that proteins cope with external stress by adapting their structure to form different ensembles of conformational substates. These states are distinguished by their activation enthalpy and entropy barriers, which can be strongly negative in the folding direction. Based on our analysis of activation compressibility and heat capacity, hydration and packing defects of the kinetic transition states are also very important for determining the reaction path. We expect that a more generalized use of this experimental approach should allow researchers to obtain greater insight into the mechanisms of physiologically relevant protein structural changes.

Long-term Outcome of Renal Transplantation in Patients with Congenital Lower Urinary Tract Malformations: A Multicenter Study.

BACKGROUND: Renal insufficiency can occur in patients with congenital lower urinary tract malformations (LUTM) even when managed during infancy. Data in the current literature concerning this subject remain sparse. The aim of this study was to report the feasibility and long-term results of renal transplantation during adulthood in patients with a congenital LUTM. METHODS: A retrospective multicenter study from 3 French renal transplant centers was conducted, including 123 transplantations on 112 patients with LUTM (1996-2016). Graft survival, patient survival, and complications were analyzed. Results were stratified according to the underlying uropathy and the type of initial management during childhood or before transplantation. RESULTS: In this study, patients suffering from posterior urethral valves (n = 49), spina bifida (n = 21), central neurogenic bladder (n = 13), bladder exstrophy (n = 14), prune belly syndrome (n = 12), Hinman syndrome (n = 6), urogenital sinus (n = 4), and other pathologies (n = 4) were included. The mean age at transplantation was 32.1 years old (±11.2). The mean follow-up period was 7.2 years. Patient survival at 1, 5, 10, and 15 years was 97.4%, 93.0%, 89.4%, and 80.0%, respectively. Graft survival at 1, 5, 10, 15, and 20 years was 96.6%, 87.6%, 77.3%, 60.6%, and 36.4%, respectively. Enterocystoplasty and continent urinary diversions exposed grafts to more frequent acute pyelonephritis (P = 0.02). There was no difference in graft survival when transplantation was performed on an enterocystoplasty or urinary diversions compared with a native bladder, provided a well-conducted bladder management. CONCLUSIONS: Even though enterocystoplasty and continent urinary diversions exposed grafts to more frequent acute graft pyelonephritis, patient and graft survival rates in LUTM at 10 years were similar to other kidney transplantations on native bladders.

Pressure effects on the structure and stability of the hyperthermophilic trehalose/maltose-binding protein from Thermococcus litoralis.

In this work, we investigated the effect of pressure on the structure and stability of the recombinant D-trehalose/D-maltose-binding protein isolated from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). The spectroscopic results obtained both in the absence and in the presence of maltose or trehalose revealed that the TMBP-Mal complex exhibits a larger structural stability under high pressure values than TMBP-Tre complex. In addition, the results also pointed out that pressure induces reversible denaturation transitions of the protein structure. By combining the fluorescence results obtained with 8-anilino-1-naphtalene sulfonate as extrinsic probe and the intrinsic indolic fluorescence of TMBP, it is evident that the protein structural changes above 400 MPa that involve the exposure to the solvent of a large portion of the hydrophobic protein domains are preceded by a partially unfolded protein structural state. The spectroscopic results have been interpreted and discussed by taking into account the X-ray structure of the protein and, in particular, the interactions of maltose and trehalose within the three-dimensional structure of TMBP.

Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps.

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (DeltaH(#)), and activation entropy (DeltaS(#)) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative DeltaH(#) of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro(114) isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the beta-hairpin comprising Trp(115). A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.

Pressure and temperature jump relaxation kinetics of the conformational change in Salmonella typhimurium tryptophan synthase L-serine complex: large activation compressibility and heat capacity changes demonstrate the contribution of solvation.

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.

Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase.

Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase alpha2beta2 multienzyme complex. We have measured the conformational equilibria of l-Ser and l-Trp complexes, using absorption and fluorescence spectrophotometry with hydrostatic pressure equilibrium perturbation. The effects of monovalent cations, disodium alpha-glycerophosphate (Na2GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of betaE109D and betaD305A mutations, on K(eq) for the conformational equilibria were determined. The l-Ser external aldimine-aminoacrylate equilibrium (K(eq)=[external aldimine]/[aminoacrylate]) has the largest value with Na+ (0.12), followed by K+ (0.04), Li+ (7.6 x 10(-4)), Rb+ (4.3 x 10(-4)), NH4+ (2.3 x 10(-4)), no cation (2.0 x 10(-4)) and Cs+ (1.6x10(-5)). alpha-Site ligands, Na(2)GP and IAG, have modest 3- to 40-fold effects on K(eq) in the direction of aminoacrylate, but BZI in the presence of Na+ gives a low value of K(eq) comparable to that obtained with Cs(+). There is no additivity of free energy for Na2GP and BZI, suggesting a common pathway for allosteric communications for both ligands. The values of DeltaV(o) range from -126 mL/mol for the Na+ complex to -204 mL/mol for the Na+ complex with BZI. The betaD305A mutation changes the K(eq) by a factor of at least 10(5) (26.7kJ/mol) and nearly abolishes allosteric communications. There are also dramatic decreases in the magnitude of both DeltaV(o) and DeltaS for the l-Ser external aldimine-aminoacrylate equilibrium for betaD305A Trp synthase, consistent with a large decrease in solvation accompanying the conformational change in betaD305A Trp synthase relative to wild-type Trp synthase. The betaE109D mutation has more modest but significant effects on K(eq), which differ with the ligand, ranging from 40-fold for GP to 2200-fold for BZI, even though betaGlu-109 is not directly involved in allosteric communications. The effect of GP on the external aldimine-quinonoid intermediate equilibrium of the Trp synthase-l-Trp complex is similar to that of GP on the Trp synthase-l-Ser external aldimine-aminoacrylate equilibrium. These results have allowed a quantitative comparison of the allosteric effects of ligand and mutations in Trp synthase. These allosteric effects are finely tuned to control the synthesis of l-Trp without resulting in substrate or product inhibition.

Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres.

Proteins were precipitated to ensure their stability upon subsequent encapsulation within PLGA microspheres. Spherical, nanosized protein particles were formed by the addition of a salt (sodium chloride) and a water-miscible organic solvent (glycofurol) to protein solutions. Various process parameters were modified to optimize the precipitation efficiency of four model proteins: lysozyme, alpha-chymotrypsin, peroxidase and beta-galactosidase. As monitored by enzymatic activity measurement of the rehydrated particles, conditions to obtain more than 95% of reversible precipitates were defined for each protein. The study of the structure of the rehydrated particles by absorbance spectroscopy, fluorescence spectroscopy and circular dichroism showed an absence of structural-perturbation after precipitation. Protein particles were then microencapsulated within PLGA microspheres using s/o/w technique. The average encapsulation yield was around 80% and no loss of protein activity occurred after the encapsulation step. Additionally, a lysozyme in vitro release study showed that all of the released lysozyme was biologically active. This method of protein precipitation is appropriate for the encapsulation in PLGA microspheres of various proteins without inactivation.

High-pressure studies of the reaction mechanism of nitric-oxide synthase.

Nitric-oxide synthase (NOS) generates nitric oxide from l-arginine in two reaction cycles with N(omega)-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them.

The use of pressure-jump relaxation kinetics to study protein folding landscapes.

Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.

Tetrahydrobiopterin as combined electron/proton donor in nitric oxide biosynthesis: cryogenic UV-Vis and EPR detection of reaction intermediates.

The role of tetrahydrobiopterin (BH4) as a cofactor in nitric oxide synthase (NOS) has been the object of intense research in the last few years. It was found that in addition to its established effects on the NOS heme spin state, substrate affinity, and enzyme dimerization, BH4 is required as a one-electron donor to oxyferrous [Fe(II).O2] heme that is formed as an intermediate in the catalytic cycle. Cryogenic spectroscopic techniques proved particularly useful in the identification of this role of BH4 in NO synthesis. With these methods, the mechanism of fast reactions, such as the reaction of ferrous NOS with O2, can be unraveled by lowering the reaction temperature to subzero values. This may not only reduce the rate to such an extent that the reaction can be followed on a time scale from seconds to minutes, but intermediates may be observed that do not accumulate at higher temperatures. Cryogenic ultraviolet-visible (UV-vis) and electron paramagnetic resonance spectroscopy have been applied to clarify why the BH4 analogue 4-amino-tetrahydrobiopterin (4-amino-BH4) is unable to support NO synthesis. In the course of these studies, evidence was gathered supporting a role for BH4 as an obligate proton and electron donor. It is believed that the inhibitory action of 4-amino-BH4 derives from an inability to serve as a proton donor, even though it is perfectly able to serve as an electron donor. In this chapter, the suitability, drawbacks, and advantages of cryogenic methods are discussed.