Molecular factors involved in the formation of secondary vascular tissues and lignification in higher plants

The formation of secondary vascular tissues involves complex processes and many steps, a number of which have been examined in detail in this study. A novel CuZn-SOD, with a high pI and thus denoted hipI-SOD, was identified and characterized in Pinus sylvestris. Results from immunolocalisation analyses indicated that it is localised in lignified structures, suggesting that SOD might participate in the formation of secondary cell walls and lignification. To further investigate its role in these processes, a Zinnia mesophyll cell system was set up. This enabled us to follow the differentiation from mesophyll cell to tracheary element. Various inhibitors against SOD and H2O2-production were applied. The results suggested that HipI-SOD might have a novel and important function in secondary cell wall formation and lignification processes. The expression pattern and localization of the protein during formation of tracheary elements support this assumption. The other part of this study involved analysis of transcription factors and their regulation, especially in secondary vascular tissues. The genes encoding three MYB-transcription factors and one novel Zinc-finger transcription factor were found in an EST-library from the cambial region of hybrid aspen (Populus tremula L. x tremuloides Michx.). The genes were cloned and characterized and their regulation by hormones, sucrose and gravity was investigated. The genes were found to be under hormone and sucrose control, and their expression altered during tension wood formation. Transgenic plants were constructed, carrying one of two antisense constructs of MYB-genes, PttMYB46 or PttMYB76, which were strongly expressed in lignified tissues. Analysis of plants with either of these constructs displayed a complex phenotype, including reduced growth, increased concentration of some phenolic acids and changes in lignin composition. Some of the phenotypic traits were indicative of strong investment in defensive characters

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

This work was supported in part by the European Union, Area NMP.2013.1.1–2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530, and by the EU Seventh Framework Programme (FP7 2007–2013) under the WallTraC project (Grant Agreement no 263916), and BioStruct-X (grant agreement no 283570). This paper reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein. CMGAF is also supported by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-PRO/103980/2008 and EXPL/BIA-MIC/1176/2012. EAB is also funded by a grant (No. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel and by a grant (No. 2013284) from the U.S.-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry.Peer reviewedPublisher PD

Filling the Gaps to Solve the Extensin Puzzle

Extensins (EXTs) are highly repetitive plant O-glycoproteins that require several post-translational modifications (PTMs) to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then they are O-glycosylated on hydroxyproline and serine. After secretion into the apoplast, O-glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in the identification of the enzymatic machinery required to process EXTs, which includes prolyl 4-hydroxylases, glycosyltransferases, papain-type cysteine endopeptidases, and peroxidases. EXTs are abundant in plant tissues and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fast-growing cells, although the molecular mechanisms underlying this regulation are unknown. In this review, we highlight recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in cell walls, and possible sensing mechanisms involving the Catharanthus roseus cell surface sensor receptor-like kinases located at the interface between the apoplast and the cytoplasmic side of the plasma membrane. This review describes recent progress in our understanding of extensin post-translational modifications throughout the secretory pathway, extensin secretion and assembly in the cell walls, and possible sensing mechanisms at the interface between the apoplast and the cytoplasmic side of the cell surface.Fil: Marzol, Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Borassi, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bringas, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Sede, Ana Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Rodríguez Garcia, Diana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Capece, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Estevez, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Peroxisomal Proteostasis Involves a Lon Family Protein That Functions as Protease and Chaperone

Proteins are subject to continuous quality control for optimal proteostasis. The knowledge of peroxisome quality control systems is still in its infancy. Here we show that peroxisomes contain a member of the Lon family of proteases (Pln). We show that Pln is a heptameric protein and acts as an ATP-fueled protease and chaperone. Hence, Pln is the first chaperone identified in fungal peroxisomes. In cells of a PLN deletion strain peroxisomes contain protein aggregates, a major component of which is catalase-peroxidase. We show that this enzyme is sensitive to oxidative damage. The oxidatively damaged, but not the native protein, is a substrate of the Pln protease. Cells of the pln strain contain enhanced levels of catalase-peroxidase protein but reduced catalase-peroxidase enzyme activities. Together with the observation that Pln has chaperone activity in vitro, our data suggest that catalase-peroxidase aggregates accumulate in peroxisomes of pln cells due to the combined absence of Pln protease and chaperone activities.

Diversity and function of anti-modified protein autoantibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease particularly affecting synovial joints. Anti-citrullinated protein autoantibodies (ACPA) are detected in the serum of about 2/3 of RA patients and are being used to classify the disease. These autoantibodies may occur years before any signs of arthritis which implies that they are a cause rather than a consequence of disease. By using modified autoantigens to detect ACPAs in large patient cohorts and by purifying polyclonal autoantibodies from patients, the hunt for disease promoting autoantigens has been going on for decades. However, the relationship between ACPA specificity and any functional effects remains unclear. In order to understand the evolution, specificity, and function of autoreactive B cells in RA, the focus of this thesis is the generation of monoclonal antibodies (mAbs) from paired variable heavy- and light-chain immunoglobulin (Ig) sequences from identified single B cells from various RA tissue compartments. We have identified citrulline-reactive autoantibody producing plasma cells in the synovium of RA patients with established disease. By generating single plasma cell derived mAbs, we learned that such autoantibodies may be directly involved in the pathogenesis of RA by promoting bone degrading osteoclasts. The mAbs were all multireactive to citrulline-peptides and citrullinated proteins, but with unique distinct binding patterns. We recognized glycine in +1 position to the citrulline and a fraction of the citrulline-reactive mAbs cross-reacted with carbamyl-peptides. In addition, the identified plasma cells displayed features of high somatic mutations and fragment antigen binding (Fab) variable N-glycosylation sites introduced by affinity maturation. By analyzing a selection of RA patient B cell derived mAbs for reactivity against apoptotic cells and activated neutrophils, we learned that a subset of the citrulline reactive mAbs bound nuclear antigens. Interestingly, a fraction of these mAbs could target nuclear histones independently of the citrullinating enzyme PAD by binding to acetylated histones. We explored the extent of the ACPA mAb multireactivity and cross-reactivity by acknowledging the importance of neighboring amino acids in addition to glycine in +1 to the citrulline. By analyzing the bone marrow plasma cell repertoire of RA patients, we observed differences in Ig-frequencies and variable Fab N-linked glycosylation sites between ACPA+ and ACPA- patients. We also found RA patient bone marrow plasma cell clonotypes. In addition, we identified citrulline-reactive bone marrow plasma cells that could bind activated neutrophils which strengthen previous reports of citrullinated histones as ACPA targets. Lastly, we identified autoantibodies against the oxidation-induced post translational protein modification adducts malondialdehyde (MDA) and malondialdehyde-acetaldehyde (MAA) in the bone marrow and lung of RA patients and individuals that also harbor ACPAs. A majority of these autoantibodies, that can promote osteoclastogenesis, need cross-linked MAA-protein for recognition independent of protein backbone. Taken together, the generation of RA patient single B cell derived mAbs, have revealed remarkable features of the autoreactivity that increases the understanding of B cell involvement in the pathogenesis of RA

Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: implications for B-cell selection

Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly-mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decreased in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly also through non-antigen driven mechanisms

Enzyme Inhibitors and Activators

Enzymes are very effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Enzyme inhibitors and activators that modulate the velocity of enzymatic reactions play an important role in the regulation of metabolism. Enzyme inhibitors are also useful tool for study of enzymatic reaction as well as for design of new medicine drugs. In this chapter, we focused on the properties of enzyme inhibitors and activators. Here we present canonical inhibitor classification based on their kinetic behavior and mechanism of action. We also considered enzyme inhibitors that were used for design of various types of pharmacological drugs and natural inhibitors as a plausible source for design of future drugs. Mechanisms of action of enzyme activators and some features of allosteric modulators are considered

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition

Glutathione Metabolism in Renal Cell Carcinoma Progression and Implications for Therapies

A significantly increased level of the reactive oxygen species (ROS) scavenger glutathione (GSH) has been identified as a hallmark of renal cell carcinoma (RCC). The proposed mechanism for increased GSH levels is to counteract damaging ROS to sustain the viability and growth of the malignancy. Here, we review the current knowledge about the three main RCC subtypes, namely clear cell RCC (ccRCC), papillary RCC (pRCC), and chromophobe RCC (chRCC), at the genetic, transcript, protein, and metabolite level and highlight their mutual influence on GSH metabolism. A further discussion addresses the question of how the manipulation of GSH levels can be exploited as a potential treatment strategy for RCC

Selective glucocorticoid receptor properties of GSK866 analogs with cysteine reactive warheads

Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR-and NF kappa B dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders