Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report by yeast two-hybrid, bioinformatics, functional and structural studies the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240). Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD and that both domains interact with FPC4. We have determined a 1.9 Å resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids.     

Slightly modifying pseudoproline dipeptides incorporation strategy enables solid phase synthesis of a 54 AA fragment of caveolin‐1 encompassing the intramembrane domain

This work contributes to highlight the benefits of pseudoproline dipeptides introduction in difficult SPPS. We show how a slight modification in the positioning choice conditioned the synthesis achievement of a 54 amino acid long caveolin‐1 peptide encompassing the intramembrane domain. Furthermore, we report a side reaction correlated with the coupling steps and generating truncated fragments with a mass deviation of + 42 Da. Considering the need of structural data for membrane proteins, most of which are considered as prevalent therapeutic targets, chemical synthesis provides an interesting alternative pathway to obtain hydrophobic domains by pushing back the frontiers of conventional RP methods of purification. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Design and evaluation of 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines as inhibitors of checkpoint and other kinases

A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold.     

Novel potent apoA-I peptide mimetics that stimulate cholesterol efflux and pre-β particle formation in vitro

Reverse cholesterol transport (RCT) is believed to be the primary mechanism by which HDL and its major protein apoA-I protect against atherosclerosis. Starting from the inactive 22-amino acid peptide representing the consensus sequence of the class A amphipathic helical repeats of apoA-I, we designed novel peptides able to mobilize cholesterol from macrophages in vitro, and to stimulate the formation of ‘nascent HDL’ particles, with potency comparable to the entire apoA-I protein.     

Structure–activity relationships amongst 4-position quinoline methanol antimalarials that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum

Utilizing mefloquine as a scaffold, a next generation quinoline methanol (NGQM) library was constructed to identify early lead compounds that possess biological properties consistent with the target product profile for malaria chemoprophylaxis while reducing permeability across the blood–brain barrier. The library of 200 analogs resulted in compounds that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum. Herein we report selected chemotypes and the emerging structure–activity relationship for this library of quinoline methanols.     

Evaluation of serum protein electrophoresis in greater rhea (<Emphasis Type="Italic">Rhea americana</Emphasis> Linnaeus, 1758)

Serum proteins from 47 healthy greater rheas (Rhea americana; male and female) were separated by electrophoresis in order to characterize normal reference ranges. Determination of total protein concentration was performed through biuret reaction. The mean value of total serum protein was 4.4 g/dL. Absolute concentrations of serum proteins were determined by agarose gel electrophoretic fractioning. Five fractions were analyzed: albumin, alpha, beta1, beta2, and gamma globulins, and the average values in grams per deciliter were 2.39, 0.32, 0.45, 0.30, and 0.79, respectively.     

Distribution of myogenic progenitor cells and myonuclei is altered in women with vs. those without chronically painful trapezius muscle

It is hypothesized that repeated recruitment of low-threshold motor units is an underlying cause of chronic pain in trapezius myalgia. This study investigated the distribution of satellite cells (SCs), myonuclei, and macrophages in muscle biopsies from the trapezius muscle of 42 women performing repetitive manual work, diagnosed with trapezius myalgia (MYA; 44 ± 8 yr; mean ± SD) and 20 matched healthy controls (CON; 45 ± 9 yr). Our hypothesis was that muscle of MYA, in particular type I fibers, would demonstrate higher numbers of SCs, myonuclei, and macrophages compared with CON. SCs were identified on muscle cross sections by combined immunohistochemical staining for Pax7, type I myosin, and laminin, allowing the number of SCs associated with type I and II fibers to be determined. We observed a pattern of SC distribution in MYA previously only reported for individuals above 70 yr of age. Compared with CON, MYA demonstrated 19% more SCs per fiber associated with type I fibers (MYA 0.098 ± 0.039 vs. CON 0.079 ± 0.031; P < 0.05) and 40% fewer SCs associated with type II fibers (MYA 0.047 ± 0.017 vs. CON 0.066 ± 0.035; P < 0.05). The finding of similar numbers of macrophages between the two groups was not in line with our hypothesis and suggests that the elevated SC content of MYA was not due to heightened inflammatory cell contents, but rather to provide new myonuclei. The findings of greater numbers of SCs in type I fibers of muscle subjected to repeated low-intensity work support our hypothesis and provide new insight into stimuli capable of regulating SC content.     

Oligomerization and insulin interactions of proinsulin C-peptide: Threefold relationships to properties of insulin

Three principally different sites of action have been reported for proinsulin C-peptide, at surface-mediated, intracellular, and extracellular locations. Following up on the latter, we now find that (i) mass spectrometric analyses reveal the presence of the C-peptide monomer in apparent equilibrium with a low-yield set of oligomers in weakly acidic or basic aqueous solutions, even at low peptide concentrations (sub-μM). It further shows not only C-peptide to interact with insulin oligomers (known before), but also the other way around. (ii) Polyacrylamide gel electrophoresis of C-peptide shows detectable oligomers upon Western blotting. Formation of thioflavin T positive material was also detected. (iii) Cleavage patterns of analogues are compatible with C-peptide as a substrate of insulin degrading enzyme. Combined, the results demonstrate three links with insulin properties, in a manner reminiscent of amyloidogenic peptides and their chaperons in other systems. If so, peripheral C-peptide/insulin interactions, absolute amounts of both peptides and their ratios may be relevant to consider in diabetic and associated diseases.