Translating Senotherapeutic Interventions into the Clinic with Emerging Proteomic Technologies.

Cellular senescence is a state of irreversible growth arrest with profound phenotypic changes, including the senescence-associated secretory phenotype (SASP). Senescent cell accumulation contributes to aging and many pathologies including chronic inflammation, type 2 diabetes, cancer, and neurodegeneration. Targeted removal of senescent cells in preclinical models promotes health and longevity, suggesting that the selective elimination of senescent cells is a promising therapeutic approach for mitigating a myriad of age-related pathologies in humans. However, moving senescence-targeting drugs (senotherapeutics) into the clinic will require therapeutic targets and biomarkers, fueled by an improved understanding of the complex and dynamic biology of senescent cell populations and their molecular profiles, as well as the mechanisms underlying the emergence and maintenance of senescence cells and the SASP. Advances in mass spectrometry-based proteomic technologies and workflows have the potential to address these needs. Here, we review the state of translational senescence research and how proteomic approaches have added to our knowledge of senescence biology to date. Further, we lay out a roadmap from fundamental biological discovery to the clinical translation of senotherapeutic approaches through the development and application of emerging proteomic technologies, including targeted and untargeted proteomic approaches, bottom-up and top-down methods, stability proteomics, and surfaceomics. These technologies are integral for probing the cellular composition and dynamics of senescent cells and, ultimately, the development of senotype-specific biomarkers and senotherapeutics (senolytics and senomorphics). This review aims to highlight emerging areas and applications of proteomics that will aid in exploring new senescent cell biology and the future translation of senotherapeutics.

A Catalog of Coding Sequence Variations in Salivary Proteins' Genes Occurring during Recent Human Evolution.

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins' gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5'-3' untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes.

The Post-Translational Modifications of Human Salivary Peptides and Proteins Evidenced by Top-Down Platforms.

In this review, we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. In the beginning, we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section, the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteomes. Three sections are dedicated to the description of glycation and enzymatic glycosylation. Citrullination and N- and C-terminal post-translational modifications (PTMs) and miscellaneous other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed throughout the sections of the manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.

Corrigendum: Top-Down Proteomics of Human Saliva Highlights Anti-inflammatory, Antioxidant, and Antimicrobial Defense Responses in Alzheimer Disease.

[This corrects the article DOI: 10.3389/fnins.2021.668852.].

Saliva, a bodily fluid with recognized and potential diagnostic applications.

Human whole saliva is a bodily fluid that can be obtained easily by noninvasive techniques. Specimens can be collected by the patient also at home in order to monitor health status and variations of several analytes of clinical interest. The contributions to whole saliva include secretions from salivary glands and, among others, from the gingival crevicular fluid that derives from the epithelial mucosa. Therefore, saliva is currently a relevant diagnostic fluid for many substances, including steroids, nonpeptide hormones, therapeutic drugs, and drugs of abuse. This review at first briefly describes the different contributions to whole saliva. A section illustrates the procedures for the collection, handling, and storage of salivary specimens. Another section describes the present use of whole saliva for diagnostic purposes and its specific utilization for the diagnosis of several local and systemic diseases. The final sections illustrate the future opportunities offered by various not conventional techniques with a focus on the most recent -omic investigations. It describes the various issues that have to be taken into account to avoid false positives and negatives, such as the strength of the experimental plan, the adequacy of the number of samples under study, and the proper choice of controls.

Top-Down Proteomics of Human Saliva Highlights Anti-inflammatory, Antioxidant, and Antimicrobial Defense Responses in Alzheimer Disease.

Alzheimer disease (AD) is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering the viewpoint that AD onset and worsening may be influenced by environmental factors causing infection, oxidative stress, and inflammatory reaction, we investigated the changes of the salivary proteome in a population of patients with respect to that in healthy controls (HCs). Indeed, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Moreover, the oral cavity continuously established adaptative and protective processes toward exogenous stimuli. In the present study, qualitative/quantitative variations of 56 salivary proteoforms, including post-translationally modified derivatives, have been analyzed by RP-HPLC-ESI-IT-MS and MS/MS analyses, and immunological methods were applied to validate MS results. The salivary protein profile of AD patients was characterized by significantly higher levels of some multifaceted proteins and peptides that were either specific to the oral cavity or also expressed in other body districts: (i) peptides involved in the homeostasis of the oral cavity; (ii) proteins acting as ROS/RNS scavengers and with a neuroprotective role, such as S100A8, S100A9, and their glutathionylated and nitrosylated proteoforms; cystatin B and glutathionylated and dimeric derivatives; (iii) proteins with antimicrobial activity, such as α-defensins, cystatins A and B, histatin 1, statherin, and thymosin ß4, this last with a neuroprotective role at the level of microglia. These results suggested that, in response to injured conditions, Alzheimer patients established defensive mechanisms detectable at the oral level. Data are available via ProteomeXchange with identifier PXD021538.

Trypsinogen and chymotrypsinogen: the mysterious hyper-reactivity of selected cysteines is still present after their divergent evolution.

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (KD  = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times.

HPLC-ESI-MS top-down analysis of salivary peptides of preterm newborns evidenced high activity of some exopeptidases and convertases during late fetal development.

To have information on the proteolytic activity of convertases and exo-peptidases on human salivary proteins, this study investigated the relative amounts of the truncated proteoforms in the saliva of preterm newborns and compared them with the relative amounts measured in saliva of at-term newborns, of babies (0-10 years old) and of adults. Results indicated that convertase(s), acting on acidic proline-rich proteins and histatin 3, and carboxypeptidase(s) acting on acidic proline-rich proteins, P-C peptide, histatin 6 and statherin were many folds more active in preterm newborns than in the other groups. Conversely, the aminopeptidase responsible for the removal of the N-terminal Asp residue of statherin was not active in preterm newborns, becoming active only several months after the normal term of delivery. The high activity of convertases determined in preterm newborns suggests that it is required for the molecular events connected to the fetus development, and encourages further studies devoted to the characterization of their specific substrates.

Top-Down Proteomics of Human Saliva Discloses Significant Variations of the Protein Profile in Patients with Mastocytosis.

Mastocytosis is a myeloproliferative neoplasm causing abnormal clonal mast cell accumulation in different tissues, such as skin and bone marrow. A cutaneous subtype (CM) is distinguished from a systemic one (SM); SM patients can be grouped into SM with (SM+C) or without (SM-C) additional cutaneous lesions, and their classification is often challenging. This study was purposed to highlight variations in the salivary proteome of patients with different mastocytosis subtypes and compared to healthy controls. A top-down proteomics approach coupled to a label-free quantitation revealed salivary profiles in patients different from those of controls and a down-regulation of peptides/proteins involved in the mouth homeostasis and defense, such as statherin, histatins, and acidic proline-rich proteins (aPRPs), and in innate immunity and inflammation, such as the cathepsin inhibitors, suggesting a systemic condition associated with an exacerbated inflammatory state. The up-regulation of antileukoproteinase and S100A8 suggested a protective role against the disease status. The two SM forms were distinguished by the lower levels of truncated forms of aPRPs, statherin, P-B peptide, and cystatin D and the higher levels of thymosin ß4 and α-defensins 1 and 4 in SM-C patients with respect to SM+C. Data are available via ProteomeXchange with identifier PXD017759.

Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC-High-Resolution ESI-MS/MS.

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.