Thiol redox proteomics seen with fluorescent eyes: the detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE

There is increasing evidence that several reversible oxidative post-translational modifications of protein cysteines participate in cell signalling. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes, that are collectively known as thiol redox proteomics. Recently, fluorescence derivatization methods have been developed that enable these post-translational modifications to be studied using proteomic workflows based on two-dimensional electrophoresis, which is a relatively accessible and affordable technique. As well as enabling a large number of samples to be processed, two-dimensional electrophoresis has the advantage that it does not rely on the intensive use of mass spectrometers. This methodology allows to "visualise" redox changes in a broad context and, although identification of the modified residues is not so straightforward, complementary derivatization can overcome this drawback. Here we review the different derivatization strategies that have been employed in these studies, comparing their advantages and potential limitations. We also review the applications and results obtained, with particular emphasis on those involving (patho)physiological stimuli, thereby showing the potential of these techniques to study the thiol redox proteome.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

Paracoccidioidomycosis in people living with HIV/AIDS: A historical retrospective cohort study in a national reference center for infectious diseases,Rio de Janeiro,Brazil

Paracoccidioidomycosis (PCM) is one of the main endemic systemic mycoses in Latin America, usually occurring in rural areas. When PCM occurs simultaneously with underlying immunosuppressive conditions, it can present as an opportunistic disease. Between 2000 and 2017, literature reported around 200 PCM cases in people living with HIV/AIDS (PLWHA). To address research gaps on this co-infection and to study its possible temporal changes in the last decade, we performed an active co-infection case search on the HIV/AIDS and PCM cohorts from a Brazilian reference center database from 1989 to 2019. We found 20 PLWHA among 684 PCM patients (2.92%), predominantly male (70.0%) and urban workers (80.0%). The median age of patients was higher in the 2010–2019 decade (p = 0.006). The occurrence of PCM in PLWHA was lower when compared with other fungal diseases. Although 50.0% of the patients had already been diagnosed with HIV infection and presented CD4+ T cell counts greater than 200/mm³ at the time of PCM diagnosis, the suspicion of immunosuppression in the context of atypical and more severe clinical forms of PCM revealed the diagnosis of HIV infection in 35.0% of the patients. Two (10.0%) patients had an evolution compatible with immune reconstitution inflammatory syndrome (IRIS) after starting antiretroviral therapy (ART).We highlight the importance of considering a PCM diagnosis in PLWHA to prevent a late-onset treatment and progression to severe manifestations and unfavorable outcomes. In addition, HIV investigation is recommended in PCM patients, especially those with atypical and more severe clinical presentations.     

Failure to Reject an Allografted Tumor after Elimination of Macrophages in Mice

After an i.p. transplantation of an allogeneic tumor (Meth A) to C57BL/6 mice, a macrophage (MΦ)-rich, non-T, non-NK cell population is induced as the major infiltrate and cytotoxic cells. We here evaluated the role of the MΦs in the rejection of allografted Meth A cells and characterized the MΦs in comparison with other well-known MΦs. At all time intervals after transplantation, the highest cytotoxic activities against Meth A tumor were obtained with the MΦ-rich population. In addition, the lymphocyte-rich population had a significant but low cytotoxic activity, whereas two other population types, granulocytes and large granular cells, were inactive. When the MΦ-rich or the T cell-depleted MΦ-rich population was i.p. transplanted simultaneously with Meth A cells into untreated C57BL/6 mice, the tumor cells were rejected without growth. After specific elimination of MΦs by in vivo application of dichloromethylene diphosphonate-containing liposomes, the cytotoxic activity against Meth A cells was hardly induced at the transplantation site of Meth A cells and the allografted Meth A tumor continued to grow, indicating that a type of MΦ is the effector cell essential for the rejection. In contrast to other well-known MΦs, the cytotoxic activity against Meth A cells was cell-to-cell contact dependent and soluble factor (e.g., NO and TNF-α) independent. Moreover, the cytotoxic activity of the MΦs (H-2^b) against ⁵¹Cr-labeled Meth A (H-2^d) cells was inhibited by the addition of unlabeled H-2^d, but not H-2^a, H-2^k or H-2^b, lymphoblasts as well as Meth A cells, implying the specific interaction of the MΦs with H-2^d cells.     

Construction of Novel Shuttle Vectors for Use between Moderately Halophilic Bacteria andEscherichia coli

Shuttle vectors useful for the genetic manipulation of several moderately halophilic bacteria have been constructed. These vectors are based on the minimal replicon of pCM1, a cryptic plasmid fromChromohalobacter marismortui,combined with the useful properties of pUC18 plasmid (i.e., small size, high copy number, multiple cloning sites,lacZfragment), as well as with the trimethoprim resistance gene as a selection marker for moderate halophiles. These vectors can be efficiently transferred by RP4-mediated conjugation fromEscherichia colito the moderate halophilesChromohalobacter marismortui, Deleya halophila, Halomonas elongata, Halomonas subglaciescola,andVolcaniella eurihalina.     

Molecular study of genes involved in virulence regulatory pathways in Bacillus anthracis vaccine strain "Carbosap"

This study investigated the genetic bases of attenuation in the Bacillus anthracis vaccine strain "Carbosap" used in Italy against anthrax in cattle and sheep. Twelve genes involved in virulence regulatory pathways underwent sequence analysis in comparison with a B. anthracis virulent strain.     

A genetic map of tetraploid <Emphasis Type="Italic">Paspalum notatum</Emphasis> Flügge (bahiagrass) based on single-dose molecular markers

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F₁ family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.     

Targeting EDEM protects against ER stress and improves development and survival in C. elegans

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.     

Structural basis of the lisinopril-binding specificity in N- and C-domains of human somatic ACE

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase which converts angiotensin I into the vasopressor peptide angiotensin II and also inactivates the hypotensive peptide bradykinin, playing an important role in blood pressure regulation. The present work describes the molecular modeling of the N-terminal human somatic ACE in complex with the inhibitor lisinopril, identifying the residues involved in the inhibitor-binding pocket. The obtained results identify differences in the lisinopril lysine moiety-binding residues for N- and C-terminals of sACE domains and an important carboxy-terminal proline hydrophobic accommodations mediated by the aromatic ring of Tyr532 and Tyr1128 residues, respectively. The present model will be useful for the development of a new inhibitor family based on the natural BPP peptides and derivatives, or even to improve the binding capacities and the domain specificity of the already known inhibitors.