Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation

Background

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.

Scope of review

This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.

Major conclusions

Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.

General significance

A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.     

Comparative studies on the changes of total soluble proteins and protease activity in Georgian commercial peanuts contaminated by Aspergillus flavus

The presence of Aspergillus species is an indicator of storage conditions, which also suggests the possibility of several biochemical changes in grains. A comparative change in total soluble proteins and protease activity was determined in commercial peanut seeds collected from Georgia State. Protein contents of healthy peanuts, naturally contaminated peanuts and then artificially inoculated peanut seeds with A. flavus were estimated by Bradford method, and protease activity was also determined by using the Protease Detection Kits. Protein contents and the protease activity of the peanuts varied from sample to sample. The soluble protein content of seeds was significantly higher in healthy peanuts than in artificially inoculated or naturally infected peanuts with A. flavus. Protease activity was found to be higher in artificially inoculated seeds than in either naturally infected or healthy peanuts. Level of soluble proteins in buffer extracts of contaminated seeds decreased with incubation time, and protease activity increased with incubation time. These changes may be attributed to host response due to infection, contribution by A. flavus or due to biochemical alterations that occur naturally during the transition from endosperm to seedling during incubation period.     

Characterisation of digestive protease in the rose sawfly,Arge rosae Linnaeus (Hymenoptera: Argidae)

Insect midgut proteases are excellent targets for insecticidal agents such as protease inhibitors. These inhibitors are used for producing transgenic plants, resistant to pests. For achieving this goal, it is necessary to find the nature of specific proteases and their properties for adopting possible pest management procedure. Therefore, characterisation of the enzymes in the gut of the rose sawfly, Arge rosae (Hymenoptera: Argidae), responsible for proteolysis, was performed using a range of synthetic substrates and specific inhibitors. The optimum conditions for general proteases and trypsin were achieved at pH 10. The highest activity for general proteases was obtained at a temperature of 45°C. The use of specific inhibitors and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) provided evidence to suggest that most of the proteases belonged to the serine group because of high inhibitory effect of phenyl methane sulfonyl fluoride on total proteolytic activity. Also, inhibition assays and zymogram analysis showed that metalloproteases are present in A. rosae digestive system. These results indicated that A. rosae larvae mainly used serine proteases for protein digestion, with chymotrypsin as the dominant form. The kinetic parameters of trypsin-like proteases using N-benzoyl-dl-arg-p-nitroanilide as substrate indicated that the K _m and V _max values of trypsin in the gut of the fifth instar larvae were 730 ± 17.3 μM and 456 ± 13.85 nmol min^?1 mg^?1 protein, respectively.     

Different coexpressions of arthritis-relevant genes between different body organs and different brain regions in the normal mouse population

Structural changes in different parts of the brain in rheumatoid arthritis (RA) patients have been reported. RA is not regarded as a brain disease. Body organs such as spleen and lung produce RA-relevant genes. We hypothesized that the structural changes in the brain are caused by changes of gene expression in body organs. Changes in different parts of the brain may be affected by altered gene expressions in different body organs. This study explored whether an association between gene expressions of an organ or a body part varies in different brain structures. By examining the association of the 10 most altered genes from a mouse model of spontaneous arthritis in a normal mouse population, we found two groups of gene expression patterns between five brain structures and spleen. The correlation patterns between the prefrontal cortex, nucleus accumbens, and spleen were similar, while the associations between the other three parts of the brain and spleen showed a different pattern. Among overall patterns of the associations between body organs and brain structures, spleen and lung had a similar pattern, and patterns for kidney and liver were similar. Analysis of the five additional known arthritis-relevant genes produced similar results. Analysis of 10 nonrelevant-arthritis genes did not result in a strong association of gene expression or clearly segregated patterns. Our data suggest that abnormal gene expressions in different diseased body organs may influence structural changes in different brain parts.     

LabCaS: Labeling calpain substrate cleavage sites from amino acid sequence using conditional random fields

The calpain family of Ca²⁺‐dependent cysteine proteases plays a vital role in many important biological processes which is closely related with a variety of pathological states. Activated calpains selectively cleave relevant substrates at specific cleavage sites, yielding multiple fragments that can have different functions from the intact substrate protein. Until now, our knowledge about the calpain functions and their substrate cleavage mechanisms are limited because the experimental determination and validation on calpain binding are usually laborious and expensive. In this work, we aim to develop a new computational approach (LabCaS) for accurate prediction of the calpain substrate cleavage sites from amino acid sequences. To overcome the imbalance of negative and positive samples in the machine‐learning training which have been suffered by most of the former approaches when splitting sequences into short peptides, we designed a conditional random field algorithm that can label the potential cleavage sites directly from the entire sequences. By integrating the multiple amino acid features and those derived from sequences, LabCaS achieves an accurate recognition of the cleave sites for most calpain proteins. In a jackknife test on a set of 129 benchmark proteins, LabCaS generates an AUC score 0.862. The LabCaS program is freely available at: http://www.csbio.sjtu.edu.cn/bioinf/LabCaS . Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Hybrid Oligomer of Cyclonucleotides and Deoxynucleotides. A High Anti Left-handed Double Helical DNA Structure

Abstract

It has been shown by us that oligonucleotides containing cyclonucleosides with a high anti glycosidic conformation take left-handed, single and double helical structures (S. Uesugi, J. Yano, E. Yano and M. Ikehara, J. Am. Chem. Soc. 99, 2313 (1977) and references therein). In order to see whether DNA can adopt the high anti left-handed double helical structure or not, a self-complementary hexanucleotide containing 6,2′-O-cyclocytidine (C^○), 8,2′-O-cyclo- guanosine (G^○), deoxycytidine and deoxyguanosine, C^○G^{○dCdGC ○}G^○, was synthesized. Corresponding hexanucleotide containing only cyclonucleosides, C^○G^○C^○G^○C^○G^○, was also synthesized. Their conformation was examined by UV, CD and ¹H NMR spectroscopy. C^○G^○C^○G^○C^○G^○ forms an unusually stable, left-handed duplex. Imino proton NMR spectra and the results of nuclear Overhauser effect experiments strongly suggest that C^○G^○dCdGC^○G^○ take a left-handed double helical structure where the deoxynucleoside residues are involved in hydrogen bonding and take a high anti glycosidic conformation. Thus it is revealed that DNA could form a high anti, left-handed double helix which is different from that of Z-DNA under some constrained conditions.     

Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.     

Modelling family 2 cystatins and their interaction with papain

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain–cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.