Jorge M. Romero

Intracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cord

Protein S‐nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory and neurodegenerative disorders. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S‐NO linkage in intact cells. To address this issue, we conducted chase experiments in spinal cord slices incubated with S‐nitrosoglutathione (GSNO). The results show that removal of GSNO leads to a rapid disappearance of PrSNOs (t½ ∼ 2 hr), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester. Moreover, PrSNOs are stable in the presence of the GSH depletor diethyl maleate, indicating that GSH is critical for protein denitrosylation. Inhibition of GSH‐dependent enzymes (glutathione S‐transferase, glutathione peroxidase, and glutaredoxin) and enzymes that could mediate denitrosylation (alcohol dehydrogense‐III, thioredoxin and protein disulfide isomerase) do not alter the rate of PrSNO decomposition. These findings and the lack of protein glutathionylation during the chase indicate that most proteins are denitrosylated via rapid transnitrosylation with GSH. The differences in the denitrosylation rate of individual proteins suggest the existence of additional structural factors in this process. This study is relevant to our recent discovery that PrSNOs accumulate in the central nervous system of patients with multiple sclerosis.

Extracellular S-nitrosoglutathione, but not S-nitrosocysteine or N2O3, mediates protein S-nitrosation in rat spinal cord slices*

There is evidence that protein S‐nitrosothiols (PrSNOs) accumulate in inflammatory demyelinating disorders like multiple sclerosis and experimental allergic encephalomyelitis. However, very little is known regarding the mechanism by which PrSNOs are formed in target cells. The present study compares the ability of potential intercellular mediators of nitrosative damage including S‐nitrosoglutathione (GSNO), S‐nitrosocysteine and N2O3 to induce protein S‐nitros(yl)ation in the spinal cord, a CNS region that is commonly affected in multiple sclerosis and experimental allergic encephalomyelitis. The results clearly demonstrate that while all three NO‐donors cause S‐nitrosation of proteins in cell‐free systems, only GSNO is a viable S‐nitrosating agent in rat spinal cord slices. Generation of PrSNOs with GSNO occurs by S‐transnitrosation as the process was not inhibited by either the NO‐scavenger rutin or the N2O3‐scavenger azide. Contrary to other cell types, nerve cells incorporate intact GSNO and neither functional l‐amino acid transporters nor cell‐surface thiols are required. We also found that there is a restricted number of proteins available for S‐nitrosation, even at high, non‐physiological concentrations of GSNO. These proteins are highly concentrated in mitochondria and mitochondria‐rich subcellular compartments. This study is relevant to those CNS disorders characterized by excessive nitric oxide production.

S-Nitrosylation of NF-κB p65 Inhibits TSH-Induced Na+/I− Symporter Expression

Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide variety of cellular physiological processes. In thyroid cells, NO-synthase III-endogenously produced NO reduces TSH-stimulated thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Further studies indicate that NO induces thyroid dedifferentiation, because NO donors repress TSH-stimulated iodide (I(-)) uptake. Here, we investigated the molecular mechanism underlying the NO-inhibited Na(+)/I(-) symporter (NIS)-mediated I(-) uptake in thyroid cells. We showed that NO donors reduce I(-) uptake in a concentration-dependent manner, which correlates with decreased NIS protein expression. NO-reduced I(-) uptake results from transcriptional repression of NIS gene rather than posttranslational modifications reducing functional NIS expression at the plasma membrane. We observed that NO donors repress TSH-induced NIS gene expression by reducing the transcriptional activity of the nuclear factor-κB subunit p65. NO-promoted p65 S-nitrosylation reduces p65-mediated transactivation of the NIS promoter in response to TSH stimulation. Overall, our data are consistent with the notion that NO plays a role as an inhibitory signal to counterbalance TSH-stimulated nuclear factor-κB activation, thus modulating thyroid hormone biosynthesis.

Mechanisms of monomeric and dimeric glycogenin autoglucosylation.

Background: Glycogenin autoglucosylation, required to prime glycogen glucopolymerization, can be produced by the monomeric and dimeric forms of the enzyme. Results: Glycogenin intramonomer glucosylation produced full autoglucopolymerization, and intrasubunit glucosylation was necessary to complete dimer autoglucosylation. Conclusion: Glycogenin dimerization is not required for full autoglucosylation. Significance: De novo glycogen biosynthesis can be sustained by monomeric glycogenin. Initiation of glucose polymerization by glycogenin autoglucosylation at Tyr-194 is required to prime de novo biosynthesis of glycogen. It has been proposed that the synthesis of the primer proceeds by intersubunit glucosylation of dimeric glycogenin, even though it has not been demonstrated that this mechanism is responsible for the described polymerization extent of 12 glucoses produced by the dimer. We reported previously the intramonomer glucosylation capability of glycogenin without determining the extent of autoglucopolymerization. Here, we show that the maximum specific autoglucosylation extent (MSAE) produced by the non-glucosylated glycogenin monomer is 13.3 ± 1.9 glucose units, similar to the 12.5 ± 1.4 glucose units measured for the dimer. The mechanism and capacity of the dimeric enzyme to carry out full glucopolymerization were also evaluated by construction of heterodimers able to glucosylate exclusively by intrasubunit or intersubunit reaction mechanisms. The MSAE of non-glucosylated glycogenin produced by dimer intrasubunit glucosylation was 16% of that produced by the monomer. However, partially glucosylated glycogenin was able to almost complete its autoglucosylation by the dimer intrasubunit mechanism. The MSAE produced by heterodimer intersubunit glucosylation was 60% of that produced by the wild-type dimer. We conclude that both intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation. The full glucopolymerization capacity of monomeric glycogenin indicates that the enzyme is able to synthesize the glycogen primer without the need for prior dimerization.

Evidence for glycogenin autoglucosylation cessation by inaccessibility of the acquired maltosaccharide.

Glycogenin initiates the biosynthesis of proteoglycogen, the mammalian glycogenin-bound glycogen, by intramolecular autoglucosylation. The incubation of glycogenin with UDP-glucose results in formation of a tyrosine-bound maltosaccharide, reaching maximum polymerization degree of 13 glucose units at cessation of the reaction. No exhaustion of the substrate donor occurred at the autoglucosylation end and the full autoglucosylated enzyme continued catalytically active for transglucosylation of the alternative substrate dodecyl-maltose. Even the autoglucosylation cessation once glycogenin acquired a mature maltosaccharide moiety, proteoglycogen and glycogenin species ranging rM 47-200kDa, derived from proteoglycogen, showed to be autoglucosylable. The results describe for the first time the ability of polysaccharide-bound glycogenin for intramolecular autoglucosylation, providing evidence for cessation of the glucose polymerization initiated into the tyrosine residue, by inaccessibility of the acquired maltosaccharide moiety to further autoglucosylation.

C-chain-bound glycogenin is released from proteoglycogen by isoamylase and is able to autoglucosylate

Proteoglycogen glycogenin is linked to the glucose residue of the C-chain reducing end of glycogen. We describe for the first time the release by isoamylase and isolation of C-chain-bound glycogenin (C-glycogenin) from proteoglycogen. The treatment of proteoglycogen with alpha-amylase releases monoglucosylated and diglucosylated glycogenin (a-glycogenin) which is able to autoglucosylate. It had been described that isoamylase splits the glucose-glycogenin linkage of fully autoglucosylated glycogenin previously digested with trypsin, releasing the maltosaccharide moiety. It was also described that carbohydrate-free apo-glycogenin shows higher mobility in SDS-PAGE and twice the autoglucosylation capacity of partly glucosylated glycogenin. On the contrary, we found that the C-glycogenin released from proteoglycogen by isoamylolysis shows lower mobility in SDS-PAGE and about half the autoglucosylation acceptor capacity of the partly glucosylated a-glycogenin. This behavior is consistent with the release of maltosaccharide-bound glycogenin instead of apo-glycogenin. No label was split from auto-[14C]glucosylated C-glycogenin or fully auto-[14C]glucosylated a-glycogenin subjected to isoamylolysis without previous trypsinolysis, thus proving no hydrolysis of the maltosaccharide-tyrosine linkage. The ability of C-glycogenin for autoglucosylation would indicate that the size of the C-chain is lower than the average length of the other glycogen chains.

Enhancement by GOSPEL protein of GAPDH aggregation induced by nitric oxide donor and its inhibition by NAD

Glyceraldehyde‐3‐phosphate dehydrogenases (GAPDHs) competitor of Siah Protein Enhances Life (GOSPEL) is the protein that competes with Siah1 for binding to GAPDH under NO‐induced stress conditions preventing Siah1‐bound GAPDH nuclear translocation and subsequent apoptosis. Under these conditions, GAPDH may also form amyloid‐like aggregates proposed to be involved in cell death. Here, we report the in vitro enhancement by GOSPEL of NO‐induced GAPDH aggregation resulting in the formation GOSPEL‐GAPDH co‐aggregates with some amyloid‐like properties. Our findings suggest a new function for GOSPEL, contrasting with its helpful role against the apoptotic nuclear translocation of GAPDH. NAD+ inhibited both GAPDH aggregation and co‐aggregation with GOSPEL, a hitherto undescribed effect of the coenzyme against the consequences of oxidative stress.

Structural and biochemical insight into glycogenin inactivation by the glycogenosis-causing T82M mutation.

The X‐ray structure of rabbit glycogenin containing the T82M (T83M according to previous authors amino acid numbering [1]) mutation causing glycogenosis showed the loss of Thr82 hydrogen bond to Asp162, the residue involved in the activation step of the glucose transfer reaction mechanism. Autoglucosylation, maltoside transglucosylation and UDP‐glucose hydrolyzing activities were abolished even though affinity and interactions with UDP‐glucose and positioning of Tyr194 acceptor were conserved. Substitution of Thr82 for serine but not for valine restored the maximum extent of autoglucosylation as well as transglucosylation and UDP‐glucose hydrolysis rate. Results provided evidence sustaining the essential role of the lost single hydrogen bond for UDP‐glucose activation leading to glycogenin‐bound glycogen primer synthesis.

Characterization of human triosephosphate isomerase S-nitrosylation.

Triosephosphate isomerase (TPI), the glycolytic enzyme that catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), has been frequently identified as a target of S-nitrosylation by proteomic studies. However, the effect of S-nitrosylation on its activity has only been explored in plants and algae. Here, we describe the in vitro S-nitrosylation of human TPI (hTPI), and the effect of the modification on its enzymatic parameters. NO-incorporation into the enzyme cysteine residues occurred by a time-dependent S-transnitrosylation from both, S-nitrosocysteine (CySNO) and S-nitrosoglutathione (GSNO), with CySNO being the more efficient NO-donor. Both X-ray crystal structure and mass spectrometry analyses showed that only Cys217 was S-nitrosylated. hTPI S-nitrosylation produced a 30% inhibition of the Vmax of the DHAP conversion to G3P, without affecting the Km for DHAP. This is the first study describing features of human TPI S-nitrosylation.

Radiation diagnostics and dosimetry modeling for characterization of plasma-beam interactions and x-ray production for a 500kV MILO

The University of New Mexico Electrical Engineering Department has designed and fabricated a Magnetically Insulated Transmission Line Oscillator (MILO) to operate with a 100 ns to 300 ns pulse capability at 500 kV driven by a marx capacitor bank. Its main purpose is to study the interaction between beams and plasmas as they relate to high power microwave production. The coaxial geometry MILO utilizes the self-generated magnetic insulation derived from high electron currents to maintain power flow. This eliminates the need for externally applied magnetic fields. The device the n utilizes a slow wave structure to couple electron energies in to high power microwaves on the order of 1.1 GHz. The operation of the device also results in the production of ionizing bremsstrahlung radiation due to electrons impinging on the aluminum walls of the device. The number of bremsstrahlung photons, the energy of the bremsstrahlung, and the angular distribution of those photons can be analyzed to effectively postulate the behavior of the beam and plasmas evolving from the cathode and anode. This paper presents the design of a tungsten pinhole camera built to identify time inte grated electron impact and x-ray production resulting from firing the MILO; these data will then be correlated with other diagnostics and particle in cell models to gain understanding of the beam plasma interactions within the vacuum chamber. MCNP radiation transport simulations were used in conjunction with measurements taken from thermo luminescent dosimeters and pin-diode radiation detectors in order to determine the radiation dose rates within the laboratory space housing the MILO. This data was then analyzed and used to properly to design adequate shielding as well as safety procedures within the laboratory to eliminate any risk of exposure to radiation exceeding the dose limits outlined in NRC 10CFR20 and university guidelines.