Isg15 controls p53 stability and functions

Degradation of p53 is a cornerstone in the control of its functions as a tumor suppressor. This process is attributed to ubiquitin-dependent modification of p53. In addition to polyubiquitination, we found that p53 is targeted for degradation through ISGylation. Isg15, a ubiquitin-like protein, covalently modifies p53 at 2 sites in the N and C terminus, and ISGylated p53 can be degraded by the 20S proteasome. ISGylation primarily targets a misfolded, dominant-negative p53, and Isg15 deletion in normal cells results in suppression of p53 activity and functions. We propose that Isg15-dependent degradation of p53 represents an alternative mechanism of controlling p53 protein levels, and, thus, it is an attractive pathway for drug discovery.     

Distinguishing Closely Related Amyloid Precursors Using an RNA Aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β₂-microglobulin (hβ₂m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ₂m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ₂m with an EC₅₀ of ∼200 nm. NMR spectroscopy was used to assign the ¹H-¹⁵N HSQC spectrum of the B6-hβ₂m complex, revealing that the aptamer binds to the face of hβ₂m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ₂m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.     

Life history traits associated with body size covary along a latitudinal gradient in a generalist grasshopper

Animal body size often varies systematically along latitudinal gradients, where individuals are either larger or smaller with varying season length. This study examines ecotypic responses by the generalist grasshopper Melanoplus femurrubrum (Orthoptera: Acrididae) in body size and covarying, physiologically based life history traits along a latitudinal gradient with respect to seasonality and energetics. The latitudinal compensation hypothesis predicts that smaller body size occurs in colder sites when populations must compensate for time constraints due to short seasons. Shorter season length requires faster developmental and growth rates to complete life cycles in one season. Using a common garden experimental design under laboratory conditions, we examined how grasshopper body size, consumption, developmental time, growth rate and metabolism varied among populations collected along an extended latitudinal gradient. When reared at the same temperature in the lab, individuals from northern populations were smaller, developed more rapidly, and showed higher growth rates, as expected for adaptations to shorter and generally cooler growing seasons. Temperature-dependent, whole organism metabolic rate scaled positively with body size and was lower at northern sites, but mass-specific standard metabolic rate did not differ among sites. Total food consumption varied positively with body size, but northern populations exhibited a higher mass-specific consumption rate. Overall, compensatory life history responses corresponded with key predictions of the latitudinal compensation hypothesis in response to season length.     

X-ray Crystal Structure and Specificity of the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.     

The Quality of Methods Reporting in Parasitology Experiments

There is a growing concern both inside and outside the scientific community over the lack of reproducibility of experiments. The depth and detail of reported methods are critical to the reproducibility of findings, but also for making it possible to compare and integrate data from different studies. In this study, we evaluated in detail the methods reporting in a comprehensive set of trypanosomiasis experiments that should enable valid reproduction, integration and comparison of research findings. We evaluated a subset of other parasitic (Leishmania, Toxoplasma, Plasmodium, Trichuris and Schistosoma) and non-parasitic (Mycobacterium) experimental infections in order to compare the quality of method reporting more generally. A systematic review using PubMed (2000–2012) of all publications describing gene expression in cells and animals infected with Trypanosoma spp was undertaken based on PRISMA guidelines; 23 papers were identified and included. We defined a checklist of essential parameters that should be reported and have scored the number of those parameters that are reported for each publication. Bibliometric parameters (impact factor, citations and h-index) were used to look for association between Journal and Author status and the quality of method reporting. Trichuriasis experiments achieved the highest scores and included the only paper to score 100% in all criteria. The mean of scores achieved by Trypanosoma articles through the checklist was 65.5% (range 32–90%). Bibliometric parameters were not correlated with the quality of method reporting (Spearman''s rank correlation coefficient <−0.5; p>0.05). Our results indicate that the quality of methods reporting in experimental parasitology is a cause for concern and it has not improved over time, despite there being evidence that most of the assessed parameters do influence the results. We propose that our set of parameters be used as guidelines to improve the quality of the reporting of experimental infection models as a pre-requisite for integrating and comparing sets of data.     

Anti-tubercular activity and molecular docking studies of indolizine derivatives targeting mycobacterial InhA enzyme

A series of 1,2,3-trisubstituted indolizines (2a–2f, 3a–3d, and 4a–4c) were screened for in vitro whole-cell anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 2b–2d, 3a–3d, and 4a–4c were active against the H37Rv-MTB strain with minimum inhibitory concentration (MIC) ranging from 4 to 32 µg/mL, whereas the indolizines 4a–4c, with ethyl ester group at the 4-position of the benzoyl ring also exhibited anti-MDR-MTB activity (MIC = 16–64 µg/mL). In silico docking study revealed the enoyl-acyl carrier protein reductase (InhA) and anthranilate phosphoribosyltransferase as potential molecular targets for the indolizines. The X-ray diffraction analysis of the compound 4b was also carried out. Further, a safety study (in silico and in vitro) demonstrated no toxicity for these compounds. Thus, the indolizines warrant further development and may represent a novel promising class of InhA inhibitors and multi-targeting agents to combat drug-sensitive and drug-resistant MTB strains.     

β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

DNA accelerates the inhibition of human cathepsin V by serpins

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.