Armando Caseiro

Finding new posttranslational modifications in salivary proline‐rich proteins

Proline‐rich proteins (PRPs) are the most complex family of salivary peptides with distinct isoforms and PTMs. Up to date, only the serine phosphorylation at positions 8, 17, and 22 have been experimentally observed on acidic PRP (aPRPs), and at position 8 on basic PRP1 and 2. The presence of a glucoronyl group at Ser17 was also noticed on aPRP. The main goal of this study was to identify new PTMs and distinct isoforms of salivary PRPs using LC‐MALDI‐TOF/TOF. Through the salivary peptidome characterization of 20 different subjects from Control, Diabetic, and Head and Neck Cancer groups, it was possible to identify the following species: (i) N‐glycosylation sites: two in basic proline‐rich protein 2 (bPRP2), one in bPRP3 and one in bPRP4; (ii) O‐glycosylation sites: two in bPRP2 and one in aPRP; (iii) other terminal monosaccharide sites: six in bPRP1, two in bPRP2 and two in bPRP3; (iv) other modifications such as N‐terminal pyro‐Glu (two in bPRP1, six in bPRP2, eight in bPRP3 and nine in bPRP4); (v) phosphorylation in serine, three in bPRP1, one in bPRP2, one in bPRP3 and one in aPRP1; (vi) bPRP1 (allele S, allele M and variant CP5) and bPRP4 (allele M). In summary, salivary peptidome data analysis allowed the identification of 45 new PRP‐modified residues, mainly due to glycosylation, phosphorylation and conversion of Gln to pyro‐Glu. Moreover, comparing all subject groups, it was noticed a predominance of N‐acetyl hexosamine modification on bPRPs in the Head and Neck Cancer patients.

Salivary Proteome and Peptidome Profiling in Type 1 Diabetes Mellitus Using a Quantitative Approach

In the present study, we applied iTRAQ-based quantitative approach to explore the salivary proteome and peptidome profile in selected subjects with type 1 diabetes, with and without microvascular complications, aiming to identify disease-related markers. From a total of 434 distinct proteins, bactericidal/permeability-increasing protein-like 1 and pancreatic adenocarcinoma up-regulated factor were found in higher levels in the saliva of all patients while increased content of other proteins like alpha-2-macroglobulin, defensin alpha 3 neutrophil-specific, leukocyte elastase inhibitor, matrix metalloproteinase-9, neutrophil elastase, plastin-2, protein S100-A8 and protein S100-A9 were related with microvascular complications as retinopathy and nephropathy. Protein-protein interaction network analysis suggests the functional clusters defense, inflammation and response to wounding as the most significantly associated with type 1 diabetes pathogenesis. Peptidome data not only support a diabetes-related higher susceptibility of salivary proteins to proteolysis (mainly of aPRP, bPRP1 and bPRP2), but also evidenced an increased content of some specific protein fragments known to be related with bacterial attachment and the accumulation of phosphopeptides involved in tooth protection. Overall, the salivary protein and peptide profile highlights the importance of the innate immune system in the pathogenesis of type 1 diabetes mellitus and related complications. This study provides an integrated perspective of salivary proteome and peptidome that should be further explored in future studies targeting specific disease markers.

Salivary peptidome in type 1 diabetes mellitus

Diabetic patients show a high susceptibility to oral diseases of inflammatory, catabolic and chronic nature with potential impact on saliva composition. In this study, our purpose was to characterize type 1 diabetes-induced alterations in the salivary peptidome aiming to find prospective biomarkers for type 1 diabetes oral health evaluation. Peptidomic analysis of saliva from controls (n = 5) and type 1 diabetic patients (n = 5) were performed by liquid chromatography followed by mass spectrometry. The proteolytic activity and metalloproteinases expression was accessed by zymography and slot blot analysis, respectively. Data evidenced a significant increase in the percentage of peptides in diabetic patients paralleled by a higher proteolytic activity, compared with healthy individuals. The nonsalivary gland protein fragments identified in saliva were mainly derived from collagen and extracellular matrix proteins, namely collagen type I. The cleavage site frequency analysis showed significant differences between healthy and type 1 diabetic individuals, highlighting the activity of proteases such as matrix metalloproteinase-9 and cathepsin D. Our results highlight salivary collagen fragments as potential biomarkers to follow up diabetes-related oral damage.

Glutathione in multiple sclerosis.

Abstract Multiple sclerosis is a chronic inflammatory disease of the central nervous system, characterised mainly as an autoimmune neurodegenerative disorder. Its cause is unknown but multifactorial; however, some studies suggest that oxidative stress may be one of the sources, or a consequence of the disease, from loss of oxidant/antioxidant balance. This review studies glutathione, one of the most important agents of the endogenous antioxidant defence system, protecting cells from damage caused by oxidative stress. It evaluates glutathione and the enzymes glutathione peroxidase and glutathione reductase in various forms and stages of the disease. Analysis of a literature search suggests that the scientific community is not unanimous in its views, so more studies are required of patients with different forms of the disease and its manifestations, taking into account that the body functions as a whole and reacts in a compensatory manner. It would seem imperative to achieve a consensus on the pathogenesis responsible for severe disability, and explore sensitive biomarkers of its progression and indicators of oxidative stress. It is also important to promote the development of new therapies, with more studies on other substances such as acrolein, lipoic acid and dimethyl fumarate. Clarification of the mechanisms involved in oxidative stress, in different forms of multiple sclerosis, could result in improvements in the monitoring and prognosis of the disease, with subsequent increases in a patient’s quality of life.

Protease profiling of different biofluids in type 1 diabetes mellitus

OBJECTIVES We aimed to disclose the proteolytic events underlying type 1 diabetes and related complication through protease profiling in the bodily fluids serum, urine and saliva. DESIGN AND METHODS Zymography followed by LC-MS/MS was performed for protease identification and quantitative comparison of proteolytic activity between healthy, type 1 diabetic patients with no complications and with retinopathy and nephropathy. Western blotting was also accomplished for MMP-9 and MMP-2 identification and expression analysis. RESULTS Only MMP-2 and MMP-9 were observed in serum with significantly increased levels and activity observed in diabetic patients. In urine and saliva other proteases besides MMPs were identified by MS and presented disease-dependent activity variations. Among these are complex MMP-9/Neutrophil gelatinase-associated lipocalin, aminopeptidase N, azurocidin and kallikrein 1 with more activity noticed in type 1 diabetes patients with nephropathy and/or retinopathy. CONCLUSION Our data highlight the usefulness of urine and saliva for the monitoring of type-1 diabetes-related proteolytic events, where aminopeptidase N, azurocidin and kallikrein 1 appear as promising screening targets for type 1 diabetes-related complications.

Impaired protein quality control system underlies mitochondrial dysfunction in skeletal muscle of streptozotocin-induced diabetic rats

Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality.

Salivary Peptidomics Targeting Clinical Applications

Abstract In the last years, efforts have been made aiming at the systematic characterization of salivary peptidome under different pathophysiological conditions to identify new and more specific diagnostic and prognostic markers and to disclose the molecular pathways modulated by diseases. More than 2000 different peptides have already been identified as well as several posttranslational modifications including phosphorylation, sulfation, glycosylation, and proteolysis. Comparative peptidome profiling using label-free or isotope-based strategies already allowed a better understanding of the physiological role in oral cavity of some of these peptides. Nevertheless, the exploitation of the proteolytic-derived fragmentation pattern holds special promise in the identification of disease-specific signatures. This article focuses on the salivary peptidomics illustrating the most used technologies and the recent achievements in the characterization of saliva peptidome under different pathophysiological conditions and highlights the future prospects and challenges in salivary peptidomics.