Metalloprotease meprin beta generates nontoxic N-terminal amyloid precursor protein fragments in vivo

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β(-/-) mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing.     

Gene dosage-dependent effects of bcl-2 expression on cellular survival and redox status

The human oncogene bcl-2 exerts protective functions in numerous models of apoptotic cell death and increased oxidative stress. We investigated the effects of inducible bcl-2 overexpression on cellular survival and redox status in dopaminergic rat pheochromocytoma PC 12 cells. Induction of high-level expression of bcl-2 in PC 12 cells resulted in generation of oxidative stress and cessation of growth by cell cycle arrest. Cell cycle arrest in bcl-2-overexpressing PC 12 cells was prevented by an inhibitor of extracellular signal-related kinase (ERK 1/2) activation. Protective effects of bcl-2 expression against L-DOPA neurotoxicity decreased with increasing amounts of bcl-2. Furthermore, high-level bcl-2 overexpression sensitized cells towards oxidative stress and glutathione depletion. Our data suggest that bcl-2 expression is beneficial only in a limited gene dosage range and that high-level expression of bcl-2 exerts potential deleterious effects.     

B7/CD28 costimulation of T cells induces a distinct proteome pattern

Effective immune strategies for the eradication of human tumors require a detailed understanding of the interaction of tumor cells with the immune system, which might lead to an optimization of T cell responses. To understand the impact of B7-mediated costimulation on T cell activation comprehensive proteome analysis of B7-primed T cell populations were performed. Using this approach we identified different classes of proteins in T cells whose expression is either elevated or reduced upon B7-1- or B7-2-mediated CD28 costimulation. The altered proteins include regulators of the cell cycle and cell proliferation, signal transducers, components of the antigen processing machinery, transporters, cytoskeletal proteins, and metabolic enzymes. A number of differentially expressed proteins are further modified by phosphorylation. Our results provide novel insights into the complexity of the CD28 costimulatory pathway of T cells and will help to identify potential targets of therapeutic interventions for modulating anti-tumor T cell activation.     

Chiral cyclopalladated complexes derived from N,N-dimethyl-1-phenethylamine with bridging bis(diphenylphosphine)ferrocene ligand as inhibitors of the cathepsin B activity and as antitumoral agents

Chiral cyclopalladated complexes derived from N,N-dimethyl-1-phenethylamine and the coordinating ligand 1,1'-bis(diphenylphosphine)ferrocene were synthesized and studied as Cathepsin B inhibitors and antitumoral agents against solid tumors. Our results revealed that the palladium compound [Pd2(C2,N-S(-)dmpa)2(mu-dppf)Cl2] (2) was able to inhibit Cathepsin B activity in a reversible fashion. This palladacycle compound binds to free cathepsin B (E) as well as to the enzyme-substrate complex (ES) with dissociation constants of KH=12+/-1 microM and alphaKH=2.4+/-0.3 microM, respectively. The application of this complex, in Walker tumor-bearing rats, resulted in 90% inhibition of the tumor growth. Subcutaneous inoculations of 10(6) tumoral cells produced solid tumors with a mass of 4.0+/-1.0 g in 12 days Walker tumor-bearing rats. However, when these animals were treated with one dose of the palladacycle compound (2.0 mg/kg), the tumoral mass was reduced to 0.3+/-0.1 g. On the other hand, the same complex (2) did not afford any protection to mice bearing the non-metastatic Ehrlich Ascites tumor treated with doses of 0.5, 5.0, and 30 mg/kg for a period of four, three and one day, respectively, beginning 72 h after tumor inoculation. Toxicological studies using mice treated with one high dose of the complex (2) (100 mg/kg) did not show any alterations in red and white blood cell morphology 14 days after the drug administration. Similar results were obtained with hepatic, kidney, and spleen tissues. The results presented in this work introduce the title cyclopalladated complexes as promising antitumoral drugs with reduced toxicity in experimental studies.     

The adaptor function of SHP-2 downstream of the prolactin receptor is required for the recruitment of p29, a substrate of SHP-2

SHP-2, a cytosolic protein tyrosine phosphatase with two SH2 domains and multiple tyrosine phosphorylation sites, contributes to signal transduction as an enzyme and/or adaptor molecule. Here we demonstrate that prolactin (PRL) stimulation of the PRL-responsive Nb2 cells, a rat lymphoma cell line, and T47D cells, a human breast cancer cell line, lead to the complex formation of SHP-2 and growth factor receptor-bound protein-2 (grb2). Using transient co-overexpression studies of the prolactin receptor (PRLR) and several tyrosine to phenylalanine mutants of SHP-2, we show that grb2 associates with SHP-2 through the C-terminal tyrosine residues of SHP-2, Y(546) and Y(584). Furthermore, in this study, we found a highly phosphorylated, 29-kDa protein (p29), a substrate of SHP-2. The recruitment of p29 to SHP-2 requires the carboxy-terminal tyrosine residues of SHP-2 (Y(546) and Y(584)). Together, our results indicate that SHP-2 may function as an adaptor molecule downstream of the PRLR and highlight a new recruitment mechanism of SHP-2 substrates.     

Role of selection versus neutral processes determining genetic variation in a small mammal along a climatic gradient in southern Africa

Empirical evidence is accumulating that pathogens drive selection and explain common patterns of high immune gene (major histocompatibility complex, MHC) polymorphism. While most previous studies have identified that selection has acted over large time scales on the MHC, there still is a paucity of information in mammal species that demonstrates how processes operate on MHC genes in extant generations. Here we investigated 439 striped mouse individuals (Rhabdomys pumilio), trapped across seven different locations along a climatic gradient in southern Africa. Data from a previous study, conducted in the same study system, revealed that gastro-intestinal nematode infections were higher in individuals from study sites located within wetter climates compared to those from drier ones. In order to improve our understanding about the role of parasite-driven selection on the MHC in contemporary generations we tested for population divergences based on seven neutral microsatellite markers and the MHC DRB exon II locus. If divergences exist, we wanted to know if they are influenced by the spatial variation in parasite pressure mediated by different climatic conditions along the study site transect. Our analysis revealed an extensive polymorphism of 249 different MHC alleles and isolation-by-distance showed significant correlations at the microsatellite loci but not at the MHC. Nematode pressure was lowest at the driest site (Fish River Canyon, Namibia) and specifically this population revealed the highest divergence between MHC and microsatellite loci. We conclude that spatial variation in parasite pressure can facilitate local immune gene adaptations and thus mediate interactions of directional and balancing selection shaping MHC polymorphism in contemporary generations.     

The IR-15N-HSQC-AP experiment: a new tool for NMR spectroscopy of paramagnetic molecules

A crucial factor for the understanding of structure-function relationships in metalloproteins is the identification of NMR signals from residues surrounding the metal cofactor. When the latter is paramagnetic, the NMR information in the proximity of the metal center may be scarce, because fast nuclear relaxation quenches signal intensity and coherence transfer efficiency. To identify residues at a short distance from a paramagnetic center, we developed a modified version of the ¹⁵N-HSQC experiment where (1) an inversion recovery filter is added prior to HSQC, (2) the INEPT period has been optimized according to fast relaxation of interested spins, (3) the inverse INEPT has been eliminated and signals acquired as antiphase doublets. The experiment has been successfully tested on a human [Fe₂S₂] protein which is involved in the biogenesis of iron-sulfur proteins. Thirteen H_N resonances, unobserved with conventional HSQC experiments, could be identified. The structural arrangement of the protein scaffold in the proximity of the Fe/S cluster is fundamental to comprehend the molecular processes responsible for the transfer of Fe/S groups in the iron-sulfur protein assembly machineries.     

Morphology of the unusual polyad in Amazonian Parkia legume trees

Key message

An unusual polyad occurs in three Parkia species, named cavitate polyad. It has an internal central space full of lipoprotein substances, contacting all pollen grains, probably aiding pollen germination.

Abstract

This study details the unusual morphology of polyads of three species of Parkia (P. multijuga Benth., P. ulei (Harms) Kuhlm., and P. pendula (Willd.) Benth. ex Walp.) and suggests functions for polyad adaptive traits that are linked to the reproductive success of the species. Polyads within the anthers of the three Parkia species were analysed by surface (scanning electron microscopy) and anatomical (light microscopy) studies. Ultrastructure and development studies were carried out for P. pendula polyads. Polyads are globose and cavitated, i.e., exhibit an internal cavity that varies in size, being more conspicuous in P. ulei and P. pendula. Other differences among species are related to the polyad size, exine ornamentation and the type of substances stored in the pollen grain. The polyad internal cavity is filled with an exudate that may be related to the pollen germination through the internal pores and/or translocation of substances from the anther loculus to the inside or vice versa. This inference is supported by the following observations: the spaces between the pollen grains in a polyad are also filled with the exudate, and the exudate inside the polyad is similar to the anther locular fluid. The morphology and substances stored within the pollen grains of Parkia polyads seem to be more related to polyad functionality and physiology than to the selective pressures exerted by different pollinators on the group.     

Faox enzymes inhibited Maillard reaction development during storage both in protein glucose model system and low lactose UHT milk

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (β-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the β-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.