After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.
Amino Acid Sequence ; Calpain/genetics/*metabolism ; Chaperonin 60/chemistry/genetics/*metabolism ; Erythrocytes/parasitology ; Humans ; Malaria, Falciparum/parasitology ; Molecular Sequence Data ; Plasmodium falciparum/chemistry/enzymology/genetics/*metabolism ; Protein Binding ; Protozoan Proteins/chemistry/genetics/*metabolism ; Sequence Alignment

OBJECTIVETo explore the mechanism of podophyllotoxin nanostructured lipid carriers (POD-NLC)-induced apoptosis of VK2/E6E7 cells mediated by endoplasmic reticulum stress (ERS).

METHODSVK2/E6E7 cells cultured in vitro were exposed to 0.125, 0.25, and 0.5 µg/ml POD-NLC or blank NLC for 24 h. The intracellular calcium concentration was measured by laser scanning confocal microscopy (LSCM), and the expression levels of GRP78, GRP94, and calpain2 mRNA and proteins in the cells were detected using RT-PCR and Western blotting.

RESULTSCompared with the control cells, the cells exposed to POD-NLC showed a concentration-dependent increase of intracellular calcium concentration (P<0.01), and the differences were statistically significant between different dose groups (P<0.05). RT-PCR and Western blotting showed that POD-NLC up-regulated GRP78, GRP94 and calpain2 mRNA and proteins expressions, which showed significant differences between blank-NLC and the control groups (P>0.05).

CONCLUSIONPOD-NLC induces apoptosis of VK2/E6E7 cells possibly by triggering the endoplasmic reticulum stress response.

Apoptosis ; Calcium ; metabolism ; Calpain ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; HSP70 Heat-Shock Proteins ; metabolism ; Heat-Shock Proteins ; metabolism ; Humans ; Membrane Proteins ; metabolism ; Nanostructures ; chemistry ; Podophyllotoxin ; chemistry ; RNA, Messenger

The limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessively inherited disease caused by a mutation of the calpain 3 gene (CAPN3), and is considered one of the most prevalent subtypes of limb-girdle muscular dystrophy (LGMD). In this study, we aimed to identify CAPN3 mutations and to characterize the phenotype of Korean patients with LGMD2A. Among 35 patients with LGMD, four patients, who showed calpain 3 deficiency on western blot analysis, were analyzed in this study. Total RNA extracted from frozen muscle tissue was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using six primer pairs covering all coding sequences of CAPN3, and direct sequencing was performed. Clinical and pathological features of the patients were also reviewed. We found four different mutations in five alleles from three patients. Of the pathogenic mutations identified, two were novel (c.2125T>C and c.2355-2357delTTC), and the others had been reported elsewhere (c.440G>C, c.1076C>T). All patients showed a high CK level with predominant proximal leg weakness, and the onset was in their childhood except for one patient. Among two novel CAPN3 mutations, one was a missense mutation (c.2125T>C [p.709Ser>Pro]), and the other was a small in-frame deletion causing omission of a single amino acid (c.2355-2357delTTC [p.786delPhe]). The clinical features of our patients were generally compatible with the characteristics of LGMD2A patients described in the previous studies.
Adolescent ; Adult ; Amino Acid Sequence ; Base Sequence ; Calpain/*genetics ; DNA Primers/chemistry ; Female ; Humans ; Korea ; Male ; Middle Aged ; Molecular Sequence Data ; Muscle Proteins/*genetics ; Muscular Dystrophies, Limb-Girdle/*genetics ; *Mutation ; Sequence Homology, Amino Acid

OBJECTIVE: To explore the eff ect of artenisiae scopariae and poriae powder (ASPD) on calpain-2 expression in liver tissue from rats with obstructive jaundice. METHODS: The rat model of obstructive jaundice was established. SD rats was divided into the control group, the obstructive jaundice group, the obstructive jaundice model plus ASPD group, the obstructive jaundice model plus saline group. Th e serum levels of TBIL, ALT, AST and other biochemical indexes were detected. The pathological changes of liver tissue were evaluated by HE staining. The calpain-2 mRNA and protein expression in liver was measured by Real-time PCR and immunohistochemistry or Western blot, respectively. RESULTS: The calpain-2 mRNA and protein expression levels were significantly up-regulated in live tissues from the rats with obstructive jaundice in a time-dependent manner. The ASPD could inhibit the calpain-2 expression in rats with obstructive jaundice concomitant with the decreased liver damage and the improved liver function, suggesting that calpain-2 was involved in endoplasmic reticulum stress-mediated cellular apoptosis and the occurrence of obstructive jaundice. CONCLUSION ASPD could be used for patients with obstructive jaundice to promote the recovery of liver function after operation and to reduce the incidence of complications, which provide a theoretical basis for the reasonable application of traditional Chinese medicine in the peroperative period.
Animals ; Apoptosis ; Artemisia ; chemistry ; Calpain ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; Jaundice, Obstructive ; enzymology ; Liver ; drug effects ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.
Alkaline Phosphatase/pharmacology ; Animal ; Brain/metabolism ; Calpain/metabolism ; Carrier Proteins/*chemistry ; Caspases/metabolism ; Cattle ; Cell-Free System ; Clathrin/*chemistry ; Electrophoresis, Polyacrylamide Gel ; Glutathione Transferase/metabolism ; Lipids/chemistry ; Membrane Proteins/*chemistry ; Phosphorylation ; Protein Binding ; Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Protein Transport ; Recombinant Proteins/chemistry/metabolism ; Reticulocytes/metabolism ; Support, Non-U.S. Gov't ; Translation, Genetic ; src Homology Domains

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Adaptor Proteins ; Animal ; Brain Chemistry ; Calpain/*metabolism ; Carrier Proteins ; Caspases/*metabolism ; Cattle ; Clathrin/*metabolism ; Coated Pits, Cell-Membrane/*metabolism ; Hydrolysis ; Membrane Proteins ; Molecular Weight ; Nerve Tissue Proteins/chemistry/*isolation & purification/metabolism ; Neurons/*chemistry ; Protein Binding ; Protein Conformation ; Recombinant Proteins/chemistry/metabolism

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Adaptor Proteins ; Animal ; Brain Chemistry ; Calpain/*metabolism ; Carrier Proteins ; Caspases/*metabolism ; Cattle ; Clathrin/*metabolism ; Coated Pits, Cell-Membrane/*metabolism ; Hydrolysis ; Membrane Proteins ; Molecular Weight ; Nerve Tissue Proteins/chemistry/*isolation & purification/metabolism ; Neurons/*chemistry ; Protein Binding ; Protein Conformation ; Recombinant Proteins/chemistry/metabolism

Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.
Animal ; Antibodies, Monoclonal ; Calpain/chemistry ; Caspases/chemistry ; Clathrin-Coated Vesicles/metabolism* ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/metabolism ; Escherichia coli/genetics ; Female ; Glutathione Transferase/genetics* ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins/metabolism ; Nerve Tissue Proteins/metabolism ; Nerve Tissue Proteins/chemistry* ; Phosphoproteins/metabolism ; Phosphoproteins/genetics ; Phosphoproteins/chemistry* ; Protein Binding ; Rabbits ; Recombinant Fusion Proteins/metabolism ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/chemistry* ; src Homology Domains

Familial amyotrophic lateral sclerosis (fALS) is caused by mutations in Cu/Zn-superoxide dismutase (SOD1), and SOD1 aggregation and calcium toxicity are involved in neuronal death. However, the effect of altered calcium homeostasis on the SOD1 aggregation is unknown. To investigate whether calcium triggers mutant SOD1 aggregation in vitro, human mutant SOD1 (G93A) was transfected into motor neuronal cell line (VSC 4.1 cells). These cells were then treated with calcium ionophore A23187 or agents that induce intracellular calcium release like cyclic ADP ribose, ryanodine or thapsigargin. A23187 was found to increase mutant SOD1 aggregation and neuronal nitric oxide synthase (nNOS) expression. Moreover, the NOS inhibitor (L-NAME) and a NO-dependent cyclic GMP cascade inhibitor (ODQ) reduced SOD1 aggregation, whereas an exogenous NO donor (GSNO) increased mutant SOD1 aggregation, which was also prevented by NOS or cGMP cascade inhibitor. Our data demonstrate that calcium-influx increases SOD1 aggregation by upregulating NO in cultured motor neuronal cells.
Amyotrophic Lateral Sclerosis/genetics/metabolism ; Animals ; Calcimycin/pharmacology ; Calcium/*metabolism ; Calpain/metabolism ; Caspase 3/metabolism ; Cell Line ; Humans ; Ionophores/pharmacology ; Motor Neurons/*metabolism ; Multiprotein Complexes ; Mutation ; Nitric Oxide/*metabolism ; Rats ; Recombinant Proteins/chemistry/genetics/metabolism ; Superoxide Dismutase/chemistry/genetics/*metabolism ; Transfection