Vitreous humor proteome: unraveling the molecular mechanisms underlying proliferative and neovascular vitreoretinal diseases.

Proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and neovascular age-related macular degeneration (nAMD) are among the leading causes of blindness. Due to the multifactorial nature of these vitreoretinal diseases, omics approaches are essential for a deeper understanding of the pathophysiologic processes underlying the evolution to a proliferative or neovascular etiology, in which patients suffer from an abrupt loss of vision. For many years, it was thought that the function of the vitreous was merely structural, supporting and protecting the surrounding ocular tissues. Proteomics studies proved that vitreous is more complex and biologically active than initially thought, and its changes reflect the physiological and pathological state of the eye. The vitreous is the scenario of a complex interplay between inflammation, fibrosis, oxidative stress, neurodegeneration, and extracellular matrix remodeling. Vitreous proteome not only reflects the pathological events that occur in the retina, but the changes in the vitreous itself play a central role in the onset and progression of vitreoretinal diseases. Therefore, this review offers an overview of the studies on the vitreous proteome that could help to elucidate some of the pathological mechanisms underlying proliferative and/or neovascular vitreoretinal diseases and to find new potential pharmaceutical targets.

Smoothing membrane protein structure determination by initial upstream stage improvements.

Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms and perform a wide range of essential biological functions. However, despite they represent the largest class of protein drug targets, a relatively small number high-resolution 3D structures have been obtained yet. Membrane protein biogenesis is more complex than that of the soluble proteins and its recombinant biosynthesis has been a major drawback, thus delaying their further structural characterization. Indeed, the major limitation in structure determination of MP is the low yield achieved in recombinant expression, usually coupled to low functionality, pinpointing the optimization target in recombinant MP research. Recently, the growing attention that have been dedicated to the upstream stage of MP bioprocesses allowed great advances, permitting the evolution of the number of MP solved structures. In this review, we analyse and discuss effective solutions and technical advances at the level of the upstream stage using prokaryotic and eukaryotic organisms foreseeing an increase in expression yields of correctly folded MP and that may facilitate the determination of their three-dimensional structure. A section on techniques used to protein quality control and further structure determination of MP is also included. Lastly, a critical assessment of major factors contributing for a good decision-making process related to the upstream stage of MP is presented.

iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment.

Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.

Performance of hydrophobic interaction ligands for human membrane-bound catechol-O-methyltransferase purification.

Despite of membrane catechol-O-methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines' O-methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl-, epoxy-, and octyl-Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH2 PO4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1-0.5, 0.25-1, and 1% of Triton X-100. On butyl media, a stepwise strategy using 375 and 0 mM NaH2PO4, followed by three elution steps at 0.25, 0.7 and 1% Triton X-100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.