Peptidyl cyclopropenones: Reversible inhibitors,irreversible inhibitors,or substrates of cysteine proteases?

Peptidyl cyclopropenones were previously introduced as selective cysteine protease reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and investigated its interaction with papain, a prototype cysteine protease. A set of kinetics, biochemical, HPLC, MS, and ¹³C‐NMR experiments revealed that the peptidyl cyclopropenone was an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma‐lactone. Thus, a single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irreversible inhibitors and alternative substrates towards enzymes of the same family.     

Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets?

Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans. Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research. These proteases have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3'). The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively. Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from the specific exclusion of substrate types. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments will bring the desired result.     

Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases.     

Palaeodiversity and formation counts: redundancy or bias?

A key question in palaeontology is whether the fossil record taken at face value is adequate to represent true patterns of diversity through time. Some methods of assessing data quality have depended on the commonly observed covariation of palaeodiversity and fossiliferous formation counts through time, based on the assumption that the count of formations containing fossils, to a greater or lesser extent, drives diversity; but what if diversity drives formations? Close study of two fossil records, early tetrapods (Devonian–Jurassic) and dinosaurs, shows how the relationship between new taxa and new fossiliferous formations varies through research time. Initially, each new find represents a new fossiliferous formation and discovery follows the ‘bonanza’ model (fossils drive formations). In unexplored parts of the world, new taxa are identified frequently in new regions/formations. Only after time, in well‐explored continents such as Europe and North America, does collecting style switch to a mix of exploration for new formations and re‐sampling of known fossiliferous formations. Data are most striking for dinosaurs, where the Triassic–Jurassic record largely comprises finds from Europe and North America, where new formation discoveries reached their half‐life in 1914. This contrasts with the Cretaceous, which is dominated by rapidly rising discoveries from regions outside Europe and North America and the formation half‐life for these ‘new’ lands is 1986, showing that 50% of new Cretaceous dinosaur‐bearing formations were identified only in the past 30 years. The relationship between dinosaur‐bearing formations and palaeodiversity then combines three signals in variable amounts, reflecting the original diversity (relative abundances of particular taxa in different formations), redundancy (new fossiliferous formations accruing because of new fossil finds) and sampling (intensity of exploration for new fossiliferous formations, and of search within already‐sampled formations). For fossil vertebrates at least, formation counts of various kinds are poor predictors of sampling, missing, for example, the bonanza samples of Lagerstätten such as the Yixian Formation in China: thousands of specimens, dozens of species, but counted as one formation. These observations suggest that formation count cannot be regarded as an unbiased metric of sampling.     

Signaling in the plant cytosol: cysteine or sulfide?

Cysteine (Cys) is the first organic compound containing reduced sulfur that is synthesized in the last stage of plant photosynthetic assimilation of sulfate. It is a very important metabolite not only because it is crucial for the structure, function and regulation of proteins but also because it is the precursor molecule of an enormous number of sulfur-containing metabolites essential for plant health and development. The biosynthesis of Cys is accomplished by the sequential reaction of serine acetyltransferase (SAT) and O-acetylserine(thiol)synthase (OASTL). In Arabidopsis thaliana, the analysis of specific mutants of members of the SAT and OASTL families has demonstrated that the cytosol is the compartment where the bulk of Cys synthesis takes place and that the cytosolic OASTL enzyme OAS-A1 is the responsible enzyme. Another member of the OASTL family is DES1, a novel l-cysteine desulfhydrase that catalyzes the desulfuration of Cys to produce sulfide, thus acting in a manner opposite to that of OAS-A1. Detailed studies of the oas-a1 and des1 null mutants have revealed the involvement of the DES1 and OAS-A1 proteins in coordinate regulation of Cys homeostasis and the generation of sulfide in the cytosol for signaling purposes. Thus, the levels of Cys in the cytosol strongly affect plant responses to both abiotic and biotic stress conditions, while sulfide specifically generated from the degradation of Cys negatively regulates autophagy induced in different situations. In conclusion, modulation of the levels of Cys and sulfide is likely critical for plant performance.     

The intermediate disturbance hypothesis--species diversity or functional diversity?

Phytoplankton dynamics in a shallow eutrophic lake were investigatedover a 3-year period with respect to environmental forces whichdrive species composition and diversity. Diversity was calculatedon the basis of species as well as on the basis of their functionalproperties (the C-R-S concept). Stratification and water columnmixing had a strong impact on phytoplankton composition. Applicationof a similarity–diversity model revealed that a high diversitywas a transient non-stable state, whereas drastic changes orlong-lasting stable environmental conditions are characterizedby low diversity. This effect was more pronounced when the diversitywas calculated on the basis of the phytoplankton species' functionalproperties. Thus, this functional approach supports the intermediatedisturbance hypothesis from field data.     

Lung cysteine cathepsins: intruders or unorthodox contributors to the kallikrein-kinin system?

The protease/antiprotease balance is tipped in favor of enhanced proteolysis in inflammatory lung disorders, promoting the spread and severity of inflammation. Cysteine cathepsins participate in the remodeling and/or degradation of the pulmonary extra cellular matrix and in lung homeostasis. There is now good evidence that cathepsins are involved in fibrosis, emphysema, asthma, and in bronchopulmonary dysplasia. Kinins are inflammatory mediators that induce edema, pain and vasodilatation, and participate in vascular homeostasis. Kinins may also contribute to the immune system by acting as danger signals, and activating bradykinin receptors. Kinins are believed to play a role in inflammatory obstructive airway diseases, asthma, and allergic rhinitis. Their release by plasma and tissue kallikreins is severely reduced at inflammatory sites, although local kinin production seems to remain intact. Such conflicting observations suggest that there are alternative mechanisms of kinin metabolism besides the classical pathways. This article reviews the biological and pathophysiological roles of lung cysteine cathepsins, kinins and their receptors, and summarizes the indications that cysteine cathepsins may contribute to kinin liberation and/or degradation.     

Truncated cystatin C in cerebrospiral fluid: Technical [corrected] artefact or biological process?

Cystatin C, a low molecular weight cysteine proteinase inhibitor present in human body fluids at physiological concentrations, is more expressed in cerebrospinal fluid (CSF) than in plasma. Mass spectrometric characterization showed that after 3 months of storage of human CSF at -20 degrees C, cystatin C was cleaved in the peptide bond between R8 and L9 and lost its eight N-termini amino acids, whereas this cleavage did not occur when stored at -80 degrees C. This truncation occurred in all CSF samples studied irrespective of the underlying neurological status, indicating a storage-related artefact rather than a physiological or pathological processing of the protein. These results stress the importance of optimal preanalytical storage conditions of any sample prior to proteomics studies.     

Desert spring refugia: museums of diversity or evolutionary cradles?

Refugia play a critical role in preserving species unable to move or adapt to cope with environmental change. The role of refugia as ‘museums of diversity’ means these environments have a high conservation priority. Less well known, however, is the role that isolated and fragmented refugia can play in the generation of new diversity. Here, we examined the diversification and evolutionary history of a community of endemic invertebrates that inhabit Australian desert spring refugia. We compared the phylogenies of seven endemic groups (Haloniscus and Phreatomerus isopods, chiltoniid amphipods, Ngarawa ostracods, Trochidrobia and Fonscochlea snails and Gymnochthebius beetles) from these springs and examine the rates and timing of diversification and reconstructed the phylogeographic history of each taxon. Despite major life‐history differences among these taxa, they demonstrate remarkable similarities in their evolutionary histories. All groups have multiple lineages that extend back to a time before the formation of present‐day deserts, and significant geographic‐based diversification since adapting to a refugial habitat. The results provide further evidence that refugia act as museums of biodiversity, preserving lineages that would have otherwise gone extinct. However, we also observed that isolation in refugia corresponds with significant diversification, leading to a recently evolved, novel endemic fauna, supporting the idea that fragmented refugia provide ideal conditions for the generation of future biodiversity hotspots.     

Functional diversity of motoneuron dendrites: by accident or design?

The distribution and geometry of the dendritic trees of spinal motoneurons obey several well-established rules. Some of these rules are based on systematic relationships between quantitative geometrical features (e.g. total dendritic length) and the three-dimensional trajectory followed by dendrites from their origin to their termination. Since dendritic geometry partially determines the transmission of current and voltage signals generated by synapses on the dendritic tree, our goal was to compare the efficacy of signal transmission by dendritic trajectories that followed different directions. To achieve this goal, we constructed detailed compartmental models of the dendritic trees of intracellularly stained neck motoneurons and calculated the electrotonic properties of each soma-to-terminal trajectory. These properties displayed a high degree of variability. To determine if this variability was due, in part, to the orientation (e.g. rostral, rostral-dorsal-lateral) of the trajectory, each trajectory was classified according to its orientation. The attenuation of current and voltage signals en route to the soma were strongly related to trajectory orientation. Trajectories with similar attenuation factors formed functional subunits that were arranged in distinct domains within the ventral horn. The difference in the efficacy of signal transmission between subunits was increased by activation of neighbouring synapses due to trajectory-related differences in non-linear summation. These results indicate that the input-output properties of motoneurons depend on the direction of the path taken by dendrites from their origin at the cell body to their terminals.     

Explaining patterns in modern ruminant diversity: contingency or constraint?

Bovid species outnumber those of cervids by over two and half to one. The present study attempts to explain this difference using two principle concepts: contingency and constraint. The concept of contingency considers the role of biogeography and the distributions and timings of major dispersals seen in the two families. Although this historical context is considered vital for understanding the problem, cervids are also characterized by their global absence from open and arid grassland habitats. This is explained in terms of phylogenetic constraint. One proposal considers the deciduous antlers of cervids as constraining the group to higher quality forage that is not generally associated with open habitats. Alternatively and more generally applicable to all ruminants is the ‘cusp fusion hypothesis’, presented here for the first time. The hypothesis seeks to explain how the primitive ruminant molar state, as seen in modern cervids and giraffids, prevents the attainment of very high molar crowns and, with it, the ability to cope with high rates of dietary tooth wear. Conversely, the derived condition of precocial cusp fusion seen in the Bovidae and Antilocapridae has enabled higher tooth crowns, their expansion into open habitats, and an increased diversity potential. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 657–672.     

Signal peptide peptidase-like 2 proteases: Regulatory switches or proteasome of the membrane?

Signal peptide peptidase-like 2 (SPPL) proteases constitute a subfamily of SPP/SPPL intramembrane proteases which are homologues of the presenilins, the catalytic core of the γ-secretase complex. The three SPPL2 proteases SPPL2a, SPPL2b and SPPL2c proteolyse single-span, type II-oriented transmembrane proteins and/or tail-anchored proteins within their hydrophobic transmembrane segments. We review recent progress in defining substrate spectra and in vivo functions of these proteases. Characterisation of the respective knockout mice has implicated SPPL2 proteases in immune cell differentiation and function, prevention of atherosclerotic plaque development and spermatogenesis. Mechanisms how substrates are selected by these enzymes are still incompletely understood. We will discuss current views on how selective SPPL2-mediated cleavage is or whether these proteases may exhibit a generalised role in the turnover of membrane proteins. This has been suggested previously for the mechanistically related γ-secretase for which the term “proteasome of the membrane” has been coined based on its broad substrate spectrum. With regard to individual substrates, potential signalling functions of the resulting cytosolic cleavage fragments remain a controversial aspect. However, it has been clearly shown that SPPL2 proteases can influence cellular signalling and membrane trafficking by controlling levels of their membrane-bound substrate proteins which highlights these enzymes as regulatory switches. Based on this, regulatory mechanisms controlling activity of SPPL2 proteases would need to be postulated, which are just beginning to emerge. These different questions, which are relevant for other families of intramembrane proteases in a similar way, will be critically discussed based on the current state of knowledge.     

Properties of metabolic graphs: biological organization or representation artifacts?

Background  

Standard graphs, where each edge links two nodes, have been extensively used to represent the connectivity of metabolic networks. It is based on this representation that properties of metabolic networks, such as hierarchical and small-world structures, have been elucidated and null models have been proposed to derive biological organization hypotheses. However, these graphs provide a simplistic model of a metabolic network's connectivity map, since metabolic reactions often involve more than two reactants. In other words, this map is better represented as a hypergraph. Consequently, a question that naturally arises in this context is whether these properties truly reflect biological organization or are merely an artifact of the representation.     

Pseudomonas corrugata: plant pathogen and/or biological resource?

Pseudomonas corrugata is the causal agent of tomato pith necrosis yet it is also used in the biological control of plant pathogenic bacteria and fungi. Potentially it could be used in other fields, such as the production of commercial biomolecules with a wide range of application and including bioremediation. This review reports the multiple characteristics of the bacterium, highlights its known molecular features and speculates on the possible underlying mechanisms of action.
Taxonomy:  Bacteria; Proteobacteria ; γ subdivision; order Pseudomonadales ; family Pseudomonadaceae ; genus Pseudomonas .
Microbiological properties:  Gram-negative, oxidase-positive, non-spore forming rods; non-fluorescent on King's B medium; produces wrinkled and rarely smooth colonies on yeast peptone glucose agar or nutrient dextrose agar; yellow to brown diffusible pigments are frequently produced.
Disease symptoms:  The typical symptom on tomato is necrosis and/or hollowing of the pith of the stem; the syndrome determines loss of turgidity of the plant, hydropic/necrotic areas and long conspicuous adventitious roots on the stem.
Biological control agent: In vitro assessed against plant pathogenic fungi and bacteria, as well as the phytotoxin indicator microorganims Rhodotorula pilimanae and Bacillus megaterium ; in vivo used against pre- and post-harvest plant pathogens.     

Oxidative stress: a theoretical model or a biological reality?

Although oxidative stress has been extensively studied the last fifteen years, many physicians and biologists are still sceptical concerning its interest in biology and medicine. This is probably due, in part, to the fact that this subject is a matter of biophysics, and the first studies reported were written using a physical language that inspired these people used to a more concrete problematic very little. Another problem is the difficulty to detect the species mediating oxidative stress, and to determine their role in biological processes. This review is aimed at presenting oxidative stress, as well as reactive oxygen species and free radicals--the molecules that mediate it--in a clear form able to convince all researchers involved in life sciences that these short-lived intermediates are indissociable from any aerobic organism. Moreover, if reactive oxygen species and free radicals are undoubtedly involved in many pathologies, they have physiological functions too.