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Biomedical subjects

M Maki

Publications and source records attributed to M Maki.

At least 19 recordsLinked to original sources
PubMed1999

Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease.

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Acetylcysteine↗
PubMed1998

Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a member of the penta-EF-hand protein family.

ALG-2 is a 22 kDa EF-hand type Ca2+-binding protein associated with lymphocyte apoptosis. Comparison of the primary structure of ALG-2 with those of EF-hand type proteins revealed that it belongs to the penta-EF-hand (PEF) protein family including the small subunit of calpain. We established a convenient method for the purification of the recombinant mouse ALG-2 expressed in Escherichia coli. The recombinant protein was first pelleted from a lysate in the absence of a Ca2+-chelator, and then extracted with buffer containing EDTA/EGTA followed by purification by conventional column chromatographies. Estimation of the molecular mass by gel filtration suggested that the recombinant ALG-2 occurred as a monomeric form. Ca2+-dependent precipitation was blocked by inclusion of non-ionic detergent Triton X-100, suggesting hydrophobic self-aggregation at high concentrations of the protein. The N-terminal deletion mutant lacking the hydrophobic non-PEF region was found to be more soluble than the wild type in the presence of Ca2+. Analysis using a fluorescent hydrophobicity probe indicated that ALG-2 exposed a hydrophobic surface in a Ca2+-concentration dependent manner, the half-maximal effect occurring at approximately 6 microM. Mg2+ was not effective for the conformational change. On Western blotting, ALG-2 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.

Amino Acid Sequence↗
PubMed1998

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

3T3 Cells↗
PubMed1998

Purification and characterization of the active-site-mutated recombinant human mu-calpain expressed in baculovirus-infected insect cells.

Recombinant human mu-calpain whose active site Cys-115 was substituted with Ser was expressed in insect cells using baculovirus system. The mutant mu-calpain, purified using an affinity-column of calpastatin oligopeptides, had no proteolytic activities of autolysis and caseinolysis. The large subunit of the mutant mu-calpain was processed from the 80 kDa form to the 76 kDa form by the wild type calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. Fluorescence polarization analysis revealed that the mutant mu-calpain retained high affinity toward fluorescein-labeled calpastatin domain 1. Fragmentation of the full-length calpastatin by the wild type calpain was enhanced by pre-incubating the inhibitor with the mutant calpain. The recombinant mutant calpain was suggested to retain the integrity of the high ordered structure of the wild type calpain.

Animals↗
PubMed1998

Calpain regulates actin remodeling during cell spreading.

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

3T3 Cells↗
PubMed1998

Run-down of the cardiac L-type Ca2+ channel: partial restoration of channel activity in cell-free patches by calpastatin.

We have found previously that run-down of cardiac Ca2+ channels in cell-free patches is reversed by cytoplasm plus adenosine triphosphate (ATP). Characterization of the factor in cytoplasm revealed that it is likely to be calpastatin (CS), an endogenous inhibitor of calpain (Ca2+-activated neutral protease). We therefore investigated the possible restoring effect of CS obtained from various tissues (activity 1.3-23 U/ml) on Ca2+ channel activity after run-down in inside-out patches. Although CS from porcine erythrocytes (plus 3 mM ATP) had only a minimal effect in restoring channel activity (to 4% of the control level recorded before the run-down), CS from porcine heart restored channel activity to 19% of control. The product of recombinant complementary deoxyribonucleic acid (cDNA) of human heart CS, a membrane-bound CS partially purified from bovine heart and CS from rabbit skeletal muscle (Sigma) restored channel activity to 28%, 23% and 10% of control levels, respectively. These results suggest that tissue-type CS, but not erythrocyte-type (truncated) CS, seems to have an effect on the cardiac Ca2+ channel to maintain its activity. Purified CS had relatively small effects compared to that of crude cytoplasm, implying that some other factor(s) might contribute also to the regulation of Ca2+ channel activity.

Adenosine Triphosphate↗
PubMed1998

Genetic susceptibility to gluten sensitive enteropathy in Irish setter dogs is not linked to the major histocompatibility complex.

Gluten sensitive enteropathy (GSE) in Irish setter dogs has been proposed as an animal model for human celiac disease (CD), in which the major histocompatibility complex (MHC) class II alleles HLA DQA1*0501 and DQB1*0201 play an important role. To investigate whether an orthologous MHC class II region is involved in canine GSE, we undertook a linkage study in two large families of gluten sensitive Irish setter dogs. A total of 44 dogs in these pedigrees were genotyped for DQA1, DQB1 and C.2202 alleles, along with 30 unrelated healthy Irish setters. No genetic linkage between the DQ or C.2002 loci and GSE was detected. In contrast to CD, susceptibility to canine GSE does not appear to be determined by variation within the MHC class II gene cluster. Therefore, canine GSE may not be an appropriate model for CD, but nevertheless remains an important disease for advancing knowledge of pathological processes in the intestine.

Alleles↗
PubMed1998

Expression of biologically active human calpastatin in baculovirus-infected insect cells and in Escherichia coli.

Calpastatin, an endogeneous inhibitor protein acting on calpain (Ca(2+)-dependent cysteine proteinase), is widely distributed in animal tissues and cells. Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Escherichia coli, were used for overexpression of the human calpastatin tagged with N-terminal hexahistidine peptide. Recombinant calpastatin was purified to homogeneity by nickel ion affinity chromatography and gel filtration separation. Purified recombinant proteins from both systems have similar inhibitory activity for calpain.

Amino Acid Sequence↗
PubMed1998

[Cardiac sympathetic denervation after aortic surgery in a patient with thoracic dissecting aneurysm: a case report].

We experienced a case of a 60 year-old man with cardiac sympathetic denervation after aortic graft replacement of ascending aorta for a dissecting aneurysm (Debakey type II). Fourteen years after pheochromocytomectomy (paraganglioma), the patient developed a severe chest pain, and admitted to the hospital for the diagnosis of dissecting aneurysm. CT scan with contrast enhancement revealed thrombosed dissecting aneurysm in the region of ascending aorta to aortic arch. Graft replacement was undergone on the same day. 123I-MIBG imaging 20 days after the operation showed severely attenuated myocardial uptake (heart to mediastinum ratio 1.19), although the MIBG imaging before the operation showed normal myocardial uptake (heart to mediastinum ratio 1.55). Heart rate variability analysis in Holter ECG showed that the power of the low frequency (LF), that of the high frequency (HF) and L/H ratio were severely decreased. MIBG and heart rate variability analysis indicated that cardiac sympathetic and parasympathetic nerve were denervated. This is the first report of cardiac sympathetic denervation after aortic vascular surgery. Clinical significance of cardiac sympathetic denervation after aortic vascular surgery is uncertain, and further investigation will be required.

3-Iodobenzylguanidine↗
PubMed1997

Regulation of cyclin D1 by calpain protease.

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.

3T3 Cells↗
PubMed1997

Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit.

The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.

Animals↗
PubMed1997

A circular dichroism study of preferential hydration and alcohol effects on a denatured protein, pig calpastatin domain I.

Pig calpastatin domain I (CSD1), a proteinase inhibitor that specifically blocks activity of calpain I and II, is a good candidate protein for studying conformational variations in the denatured form of protein. An extensive structural characterization of CSD1 reported in the first part of this work has shown that CSD1 at neutral pH is in an expanded and flexible conformation without secondary and tertiary structures. Next, we further studied cosolvent effects of protein-stabilizers, polyols and sulfate salts, as well as protein-destabilizers, alcohols, on the conformation of CSD1 monitored by far- and near-UV CD spectroscopy. We found that both groups of cosolvents at high concentration induce highly helical structures of CSD1, but without specific tertiary interactions. Based on the results on the polyols and the sulfate salts, we have suggested that the preferential hydration is one of the thermodynamic forces to induce secondary structures in the denatured state of protein. Variations in isodichroic points of changes in far-UV CD spectrum as functions of cosolvent species and their concentration have exhibited complexity of the processes. The present study implies that protein stability in the presence of cosolvents is often determined by free energy difference between the folded and the highly structured denatured state, not between the folded and the random state.

Animals↗
PubMed1997

Calpain contributes to silica-induced I kappa B-alpha degradation and nuclear factor-kappa B activation.

Both silica and lipopolysaccharide (LPS) induce a rapid degradation of I kappa B alpha, an intracellular inhibitor of the nuclear factor (NF)-kappa B transcription factor. In this report, we demonstrate that MG132, a relatively specific proteasome inhibitor, is capable of suppressing LPS-induced I kappa B alpha degradation and NF-kappa B activation in mouse macrophage line RAW 264.7 cells, but is unable to influence the same induction produced by silica. In contrast, the lysosome inhibitor chloroquine has little effect on I kappa B alpha degradation induced by either silica or LPS. In fact, chloroquine enhances the signal-induced nuclear expression of NF-kappa B p50/p65 heterodimer by inhibiting the resynthesis of I kappa B alpha. With the use of transient transfection of a plasmid that expresses calpastatin, a natural inhibitor for calpain, the silica-induced degradation of I kappa B alpha and NF-kappa B activation was attenuated. In contrast, no inhibition of LPS-induced I kappa B alpha degradation and NF-kappa B activation was observed by the overexpression of calpastatin. This suggests that calpain contributes to silica-induced I kappa B alpha degradation and NF-kappa B activation but not to LPS-induced I kappa B alpha degradation and NF-kappa B activation.

Animals↗
PubMed1997

Celiac patients predominantly inherit HLA-DPB1*0101 positive haplotype from HLA-DQ2 homozygous parent.

The DQA1*0501 and DQB1*0201 alleles (hereafter DQ2) confer genetic susceptibility to celiac disease (CD). Some studies have indicated that the DPB1, DMB, and TAP loci, that are located close to the DQ genes, could also together with DQ or independently confer genetic susceptibility to CD. Some others have claimed that these associations result merely from linkage disequilibrium, a hallmark of the MHC, that often makes the precise mapping of susceptibility genes difficult. To evaluate further the role of class II genes in CD, we analyzed segregation of DPB1 alleles in families with CD. In particular, we analyzed families where one of the parents was homozygous for the DQ2 risk allele but heterozygous for DPB1*0101, an allele claimed to be an additional risk allele. We reasoned that if DPB1*0101 would not play a role in CD, then patients should inherit the DQ2 haplotypes randomly from a homozygous parent. We here present evidence that, in all 6 informative families, those DQ2 positive haplotypes that also include the DPB1*0101 allele, rather than those without DPB1*0101, are predominantly segregated to the index patient from the parent homozygous for the DQ2 risk marker (p = 0.03 as compared to their healthy siblings). If confirmed in larger studies the results indicate that despite DQ2 other genes in or near the MHC may associate with CD.

Celiac Disease↗