CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases

Background

CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles.

Methods and results

In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at ²⁹⁶SNLD²⁹⁹ and ³⁴⁸TCPD³⁵¹, and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified ³²⁰EPRT³²³ as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at ³²⁰EPRT³²³. Additionally, the cleavage catalyzed by the ³²⁰EPRT³²³ protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment.

Conclusion

CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the “³²⁰EPRT³²³ protease(s).” Furthermore, ³²⁰EPRT³²³ cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage.

General significance

CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.     

Biosynthesis and function of chondroitin sulfate

Background

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions.

Scope of review

Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo.

Major conclusions

Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes.

General significance

Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

DISC1-related signaling pathways in adult neurogenesis of the hippocampus

Disrupted-in-schizophrenia 1 (DISC1) is a multifunctional scaffold protein which plays an important role in neurogenesis and neural development in the adult brain, especially in the dentate gyrus (DG) of the hippocampus. Accumulated research has unveiled the role of DISC1 in several aspects of neural development and neurogenesis, such as neuronal maturation, proliferation, migration, positioning, differentiation, dendritic growth, axonal outgrowth, and synaptic plasticity. Studies on the function of this protein have explored multiple facets, including variants and missense mutants in genetics, proteins interactivity and signaling pathways in molecular biology, and pathogenesis and treatment targets of major mental illness, and more. In this review, we present several signaling pathways discussed in recent research, such as the AKT signaling pathway, GABA signaling pathway, GSK3β signaling pathway, Wnt signaling pathway, and NMDA-R signaling pathway. DISC1 interacts, directly or indirectly, with these signaling pathways and they co-regulate the process of adult neurogenesis in the hippocampus.     

Phenotype and Micro-array characterization of duplication 11q22.1-q25 and review of the literature

Partial duplication of 11q is related to several malformations like growth retardation, intellectual disability, hypoplasia of corpus callosum, short nose, palate defects, cardiac, urinary tract abnormalities and neural tube defects. We have studied the clinical and molecular characteristics of a patient with severe intellectual disabilities, dysmorphic features, congenital inguinal hernia and congenital cerebral malformation which is referred to as cytogenetic exploration. We have used FISH and array CGH analysis for a better understanding of the double chromosomic aberration involving a 7p microdeletion along with a partial duplication of 11q due to adjacent segregation of a paternal reciprocal translocation t(7;11)(p22;q21) revealed after banding analysis. The patient's karyotype formula was: 46,XY,der(7)t(7;11)(p22;q21)pat. FISH study confirmed these rearrangement and array CGH technique showed precisely the loss of at least 140 Kb on chromosome7p22.3pter and 33.4 Mb on chromosome11q22.1q25. Dysmorphic features, severe intellectual disability and brain malformations could result from the 11q22.1q25 trisomy. Our study provides an additional case for better understanding and delineating the partial duplication 11q.     

Mapping and tissue mRNA expression analysis of the pig solute carrier 27A (SLC27A) multigene family

Solute-carrier family 27A molecules are integral transmembrane proteins that play a fundamental role in the uptake of long-chain fatty acids into mammalian cells. Our goal was to characterize this multigene family in pigs. Chromosomal location of the six porcine SLC27A genes was determined by radiation hybrid mapping and indicated that the six genes map to six different chromosomal locations. Moreover, we analyzed SLC27A mRNA expression in six pig tissues by quantitative RT-PCR. While SLC27A1, SLC27A3 and SLC27A4 were expressed in most, if not all, analyzed tissues, SLC27A2, SLC27A5 and SLC27A6 were predominantly expressed in the liver. In general, pig and human SLC27A mRNA expression profiles were remarkably concordant, although important differences were observed for SLC27A1 and SLC27A6 mRNAs. Discrepancies between mRNA expression profiles have been observed even in closely related primate species, and they might reflect the acquisition of regulatory changes promoting evolutionary adaptation.     

Stability of ultramer as copy number standards in real-time PCR

Since commercial copy number standards are not always available for real-time PCR, alternative sources of DNA are used. Unfortunately, stored genomic DNA or PCR amplicon has been shown to be unstable, resulting in variable copy number. More recently, the use of ultramer as copy number standard has been reported. However, there is little information on the stability of ultramer under different storage conditions. Thus the aim of this study was to determine the stability of ultramer as copy number standard under different storage conditions using different mixing methods. We found that ultramer copy number was not affected by storage at either 4 °C or − 20 °C over a period of 30 days. Furthermore, the method of mixing the ultramer did not appear to contribute to variability in results. Irrespective of storage temperature or mixing method, there was less than 5% variance in Ct value over a period of 30 days. A duplicate set of standards costs approximately $0.01. Therefore, the use of ultramer as copy number standards in real-time PCR, is cost effective and convenient.     

Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders

We reported previously that the expression of Wnt-related genes is lower in osteoporotic hip fractures than in osteoarthritis. We aimed to confirm those results by analyzing β-catenin levels and explored potential genetic and epigenetic mechanisms involved.     

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.