Margherita Ruoppolo

Folding and Oxidation of the Antibody Domain CH3

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.

Identification of tissue transglutaminase-reactive lysine residues in glyceraldehyde-3-phosphate dehydrogenase

Polyglutamine domains are excellent substrates for tissue transglutaminase resulting in the formation of cross‐links with polypeptides containing lysyl residues. This finding suggests that tissue transglutaminase may play a role in the pathology of neurodegenerative diseases associated with polyglutamine expansion. The glycolytic enzyme GAPDH previously was shown to tightly bind several proteins involved in such diseases. The present study confirms that GAPDH is an in vitro lysyl donor substrate of tissue transglutaminase. A dansylated glutamine‐containing peptide was used as probe for labeling the amino‐donor sites. SDS gel electrophoresis of a time‐course reaction mixture revealed the presence of both fluorescent GAPDH monomers and high molecular weight polymers. Western blot analysis performed using antitransglutaminase antibodies reveals that tissue transglutaminase takes part in the formation of heteropolymers. The reactive amino‐donor sites were identified using mass spectrometry. Here, we report that of the 26 lysines present in GAPDH, K191, K268, and K331 were the only amino‐donor residues modified by tissue transglutaminase.

Structural characterization of transglutaminase-catalyzed cross-linking between glyceraldehyde 3-phosphate dehydrogenase and polyglutamine repeats

The accumulation of abnormal polyglutamine‐containing protein aggregates within the cytosol and nuclei of affected neurons is a hallmark of the progressive neurodegenerative disorders caused by an elongated (CAG)n repeat in the genome. The polyglutamine domains are excellent substrates for the enzyme transglutaminase type 2 (tissue), resulting in the formation of cross‐links with polypeptides containing lysyl groups. Enzymatic activity toward the Qn domains increases greatly upon lengthening of such Qn stretches (n > 40). Among the possible amine donors, the glycolytic enzyme glyceraldehyde‐3‐phosphate‐dehydrogenase was shown to tightly bind several proteins involved in polyglutamine expansion diseases. Recently, the authors have shown that K191, K268, and K331, out of the 26 lysines present in glyceraldehyde‐3‐phosphate‐dehydrogenase, are the reactive amine‐donor sites forming cross‐links with substance P, which bears the simplest Qn domain (n = 2). The present study reports that synthetic peptides of both pathological and nonpathological length (n = 43 and 17, respectively) form cross‐links with the same K residues located in the C‐terminal region of glyceraldehyde‐3‐phosphate‐dehydrogenase. In addition, it is shown that extra K residues present in the C termini of glyceraldehyde‐3‐phosphate‐dehydrogenase are susceptible to cross‐linking in the presence of transglutaminase. The present results indicate a possible modulating effect of Qn stretches on tissue transglutaminase substrate specificity and mechanism of recognition.

Integration of Proteomics and Metabolomics in Exploring Genetic and Rare Metabolic Diseases

Background: Inherited metabolic disorders or inborn errors of metabolism are caused by deficiency of enzymatic activities in the catabolism of amino acids, carbohydrates, or lipids. These disorders include aminoacidopathies, urea cycle defects, organic acidemias, defects of oxidation of fatty acids, and lysosomal storage diseases. Inborn errors of metabolism constitute a significant proportion of genetic diseases, particularly in children; however, they are individually rare. Clinical phenotypes are very variable, some of them remain asymptomatic, others manifest metabolic decompensation in neonatal age, and others encompass mental retardation at later age. The clinical manifestation of these disorders can involve different organs and/or systems. Some disorders are easily managed if promptly diagnosed and treated, whereas in other cases neither diet, vitamin therapy, nor transplantation appears to prevent multi-organ impairment. Summary: Here, we discuss the principal challenges of metabolomics and proteomics in inherited metabolic disorders. We review the recent developments in mass spectrometry-based proteomic and metabolomic strategies. Mass spectrometry has become the most widely used platform in proteomics and metabolomics because of its ability to analyze a wide range of molecules, its optimal dynamic range, and great sensitivity. The fast measurement of a broad spectrum of metabolites in various body fluids, also collected in small samples like dried blood spots, have been facilitated by the use of mass spectrometry-based techniques. These approaches have enabled the timely diagnosis of inherited metabolic disorders, thereby facilitating early therapeutic intervention. Due to its analytical features, proteomics is suited for the basic investigation of inborn errors of metabolism. Modern approaches enable detailed functional characterization of the pathogenic biochemical processes, as achieved by quantification of proteins and identification of their regulatory chemical modifications. Key Message: Mass spectrometry-based “omics” approaches most frequently used to study the molecular mechanisms underlying inherited metabolic disorders pathophysiology are described.

FOLDING PATHWAYS OF DISULPHIDE CONTAINING PROTEINS

Protein folding, associated with oxidation and isomerization of disulphide bonds was studied using reduced and denatured RNase T1 (rd-RNase T1) or RNase A (rdRNase A) and mixed disulphide between glutathione and reduced RNase T1 (GS-RNase T1) as starting protein materials. Folding was initiated by addition of free glutathione (reduced glutathione (GSH) + oxidised glutathione (GSSG)) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process. All the analyses indicate a pathway of sequential reactions in the formation of native RNase T1 or RNase A which occurs via the reiteration of two steps: i) formation of a species containing both mixed disulphides with glutathione and free protein thiols, ii) formation of an intramolecular disulphide via thiol-disulphide interchange reaction betwen them. Refolding of rd-RNase T1 and GS-RNase T1 was also performed in the presence of Protein Disulphide Isomerase (PDI). Addition of PDI led to a catalysis of each individual reaction of the entire process without altering the refolding pathway. On the basis of these experiments an hypothesis of a general pathway for folding of S-S containing proteins is proposed.