Paulo Bastos

A glimpse into the modulation of post-translational modifications of human-colonizing bacteria.

Protein post-translational modifications (PTMs) are a key bacterial feature that holds the capability to modulate protein function and responses to environmental cues. Until recently, their role in the regulation of prokaryotic systems has been largely neglected. However, the latest developments in mass spectrometry-based proteomics have allowed an unparalleled identification and quantification of proteins and peptides that undergo PTMs in bacteria, including in species which directly or indirectly affect human health. Herein, we address this issue by carrying out the largest and most comprehensive global pooling and comparison of PTM peptides and proteins from bacterial species performed to date. Data was collected from 91 studies relating to PTM bacterial peptides or proteins identified by mass spectrometry-based methods. The present analysis revealed that there was a considerable overlap between PTMs across species, especially between acetylation and other PTMs, particularly succinylation. Phylogenetically closer species may present more overlapping phosphoproteomes, but environmental triggers also contribute to this proximity. PTMs among bacteria were found to be extremely versatile and diverse, meaning that the same protein may undergo a wide variety of different modifications across several species, but it could also suffer different modifications within the same species.

Human Antimicrobial Peptides in Bodily Fluids: Current Knowledge and Therapeutic Perspectives in the Postantibiotic Era

Antimicrobial peptides (AMPs) are an integral part of the innate immune defense mechanism of many organisms. Due to the alarming increase of resistance to antimicrobial therapeutics, a growing interest in alternative antimicrobial agents has led to the exploitation of AMPs, both synthetic and isolated from natural sources. Thus, many peptide‐based drugs have been the focus of increasing attention by many researchers not only in identifying novel AMPs, but in defining mechanisms of antimicrobial peptide activity as well. Herein, we review the available strategies for the identification of AMPs in human body fluids and their mechanism(s) of action. In addition, an overview of the distribution of AMPs across different human body fluids is provided, as well as its relation with microorganisms and infectious conditions.

Insights into the human brain proteome: Disclosing the biological meaning of protein networks in cerebrospinal fluid

Abstract Cerebrospinal fluid (CSF) is an excellent source of biological information regarding the nervous system, once it is in close contact and accurately reflects alterations in this system. Several studies have analyzed differential protein profiles of CSF samples between healthy and diseased human subjects. However, the pathophysiological mechanisms and how CSF proteins relate to diseases are still poorly known. By applying bioinformatics tools, we attempted to provide new insights on the biological and functional meaning of proteomics data envisioning the identification of putative disease biomarkers. Bioinformatics analysis of data retrieved from 99 mass spectrometry (MS)-based studies on CSF profiling highlighted 1985 differentially expressed proteins across 49 diseases. A large percentage of the modulated proteins originate from exosome vesicles, and the majority are involved in either neuronal cell growth, development, maturation, migration, or neurotransmitter-mediated cellular communication. Nevertheless, some diseases present a unique CSF proteome profile, which were critically analyzed in the present study. For instance, 48 proteins were found exclusively upregulated in the CSF of patients with Alzheimer’s disease and are mainly involved in steroid esterification and protein activation cascade processes. A higher number of exclusively upregulated proteins were found in the CSF of patients with multiple sclerosis (76 proteins) and with bacterial meningitis (70 proteins). Whereas in multiple sclerosis, these proteins are mostly involved in the regulation of RNA metabolism and apoptosis, in bacterial meningitis the exclusively upregulated proteins participate in inflammation and antibacterial humoral response, reflecting disease pathogenesis. The exploration of the contribution of exclusively upregulated proteins to disease pathogenesis will certainly help to envision potential biomarkers in the CSF for the clinical management of nervous system diseases.

Unravelling the Power of Omics for the Infertile Ageing Male

The aging phenomenon is intrinsically responsible for diseases and lifestyle-associated conditions afflicting the aging male. In particular, male infertility seems to result from deleterious lifestyle choices. However, the aging effect at the individual gene/protein level is poorly discussed and valuable information is certainly missing. We have thus used an omics approach to identify differentially expressed proteins and genes from spermatozoa and seminal plasma samples across several conditions affecting adult male fertility. Our search resulted in 400 differentially expressed proteins in seminal plasma and 409 differentially expressed proteins in spermatozoa as well as, almost 6,000 differentially expressed spermatozoa mRNAs. We have functionally analyzed these proteins and genes to understand and discuss how biological processes and signaling pathways associated with aging might affect male fertility. Sperm and seminal fluid proteins from infertile males display significant alterations in i) processes previously implicated in the aging phenomena, such as mitochondrial dysfunction, DNA instability, oxidative stress, protein misfolding and intracellular mistrafficking, and ii) processes specifically involved in reproductive phenomena, such as sperm-egg recognition/acrosome reaction, embryo and morula development, blastocyst implantation and DNA methylation involved in embryo development. These proteins display a widespread distribution and seem to be significantly influenced by deleteriously lifestyle choices. Conventional assays to assess male fertility are inadequate to comprehensively reveal the broad-spectrum of alterations at the transcriptional and translational levels afflicting the infertile aging male. In turn, proteomics and transcriptomics are suitable options for addressing these key issues that may explain many poorly understood fertility-associated phenomena resulting from the aging process.

EDTA-functionalized magnetic nanoparticles: A suitable platform for the analysis of low abundance urinary proteins

Urine is a highly attractive source of biological information and disease biomarkers, whose proteome characterization is ongoing. To that end, depletion/enrichment strategies for protein analysis can be of great convenience. We have thus developed a method based on the use of EDTA-functionalized magnetic nanoparticles (NPs@EDTA), to fractionate urine samples before liquid chromatography-mass spectrometry analysis and compared the identified proteins with those obtained from ultrafiltrated/unfractionated (UF) urine samples. NPs@EDTA allowed larger urine volumes to be processed, resulting in a greater number of protein identifications (~2-fold) at a lower cost when compared to UF samples. Proteins of greater abundance (such as albumin and uromodulin) were, at least partially, depleted with NPs@EDTA while those of lower abundance were enriched. Bioinformatics analysis showed that approximately 27% of NPs@EDTA-enriched proteins were annotated as displaying enzymatic activity, most of these being hydrolytic enzymes (56%), particularly proteases/peptidases (48%). Also, post-translational modifications were prominently predicted across NPs@EDTA-enriched proteins (90%), particularly glycosylation (52%), phosphorylation (47%) and acetylation (30%). NPs@EDTA allowed the identification of 109 proteins in urine for the first time, showing high potential as a platform for urines fractionation prior to proteomic analysis.

The Role of Urinary Proteases in Bladder Cancer

Bladder cancer (BCa) is one of the most prevalent malignancies worldwide. Risk factors for BCa are well established and include smoking and infections, which can lead to immune system activation, altered gene expression patterns, proteolytic activity, tissue damage, and, ultimately, cancer development. Urine has become one of the most attractive diagnosis samples, and, notably, urine profiling by mass spectrometry allows the simultaneously analysis of multiple enzymes and their interactors, substrates, inhibitors, and regulators, providing an integrative view of enzymatic dynamics. Most BCa-associated enzymatic alterations take place at the level of proteases, being MMP-9, MMP-2, urokinase-type plasminogen activator, cathepsin D, and cathepsin G already related to BCa development and progression. Herein, we overview the role of proteases and the classes more studied in BCa pathogenesis, as well as the methodologies used for assessing protease amount and activity in urine samples, highlighting its advantages and limitations, and the value of urinary proteases as disease biomarkers.

Methodological approaches and insights on protein aggregation in biological systems

ABSTRACT Introduction: The proper folding of native proteins is critical and dynamic, but inherently unstable. Therefore, proteins eventually end up adopting misfolded conformations which compromise their function and may even trigger aggregation. Risk factors for neurodegenerative, metabolic and heart diseases compromise cellular protein quality-control systems, promoting protein aggregation. Multiple protein post-translational modifications dynamically regulate protein aggregation and disaggregation in a very complex, intricate and delicate balance. Areas covered: Herein, we overview the more promising techniques and approaches for the elucidation of the biological implications of protein aggregation. The particular insights provided by different techniques were discriminated and several examples of post-translational modifications together with their targets were pooled and critically discussed, representing promising future therapeutic targets. Expert commentary: In the years to come, differences between physiological and pathological protein aggregation will certainly become easier to determine. Techniques such as hydrogen/deuterium exchange, circular dichroism spectroscopy and novel mass spectrometry-based approaches are being optimized and are expected to introduce inhibitors of protein aggregation into the clinic. However, protein aggregation is not an isolated phenomenon, but rather influenced by multiple cellular components which complete knowledge is still far.

A fractionation approach applying chelating magnetic nanoparticles to characterize pericardial fluid's proteome

Owing to their close proximity, pericardial fluid (PF)s proteome may mirror the pathophysiological status of the heart. Despite this diagnosis potential, the knowledge of PFs proteome is scarce. Large amounts of albumin hamper the characterization of the least abundant proteins in PF. Aiming to expand PFs proteome and to validate the technique for future applications, we have fractionated and characterized the PF, using N-(trimethoxysilylpropyl)ethylenediamine triacetic acid (EDTA)-functionalized magnetic nanoparticles (NPs@EDTA) followed by a GeLC-MS/MS approach. Similarly to an albumin-depletion kit, NPs@EDTA-based fractionation was efficient in removing albumin. Both methods displayed comparable inter-individual variability, but NPs@EDTA outperformed the former with regard to the protein dynamic range as well as to the monitoring of biological processes. Overall, 565 proteins were identified, of which 297 (>50%) have never been assigned to PF. Moreover, owing to this methods good proteome reproducibility, affordability, rapid automation and high binding ability of NP@EDTA, it bears a great potential towards future clinical application.