IS2 Region Research Articles

Separation, Purification, and Phylogenetic Characterization of Detergent-Compatible Protease Produced by Penicillium Species

Background: Filamentous fungal forms are said to secrete multiple peptidases with portent detergent capabilities in addition to their usage in food, beverages, and pharmaceutical industries. Objective: The current study isolated and purified fungal isolates from clinical samples and studied their capability as caseinolytic and in removing blood stains from fabrics. Materials and Methods: The present work isolated nine isolates, which are protease-positive and out of them eight belong to Penicillium sp. From the IS2 region, amplification of the isolates concluded their match using Blastn as Penicillium citrinum in majority. Results: The enzyme extract (P18), which was found effective in removing blood stains with good caseinolytic activity, was found to belong to P. citrinum as from Blastn studies. In addition, these proteases extracted were highly compatible with commercially available detergents. P4 was found to remove blood stains from fabrics in less than 30 min (P<0.02) when compared with other extracts (P1 and P4). Conclusion: The ability to secrete protease in promising amounts along with its potential usage as detergent makes these enzymes useful in industries, especially for the laundry industry.

The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca 2+-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca 2+. Ca 2+-dependent p94 mutants were screened using “p94-trapping”, which is an application of yeast genetic reporter system called “proteinase-trapping”. Several amino acids were revealed as critical for apparent Ca 2+-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.

Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of "idiopathic" cardiomyopathy. Recent molecular genetic analyses have now revealed that "idiopathic" cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.

Limb girdle muscular dystrophy in a sibling pair with a homozygous Ser606Leu mutation in the alternatively spliced IS2 region of calpain 3

Previous family studies revealed a large number of calpain 3 ( CAPN3 ) mutations that cause recessive forms of limb girdle muscular dystrophy (LGMD2A) with selective atrophy of the proximal limb muscles. Correlations between the nature and site of a particular mutation and its corresponding phenotype, however, can only be established from homozygous mutations, which are particularly rare in the alternatively spliced NS, IS1 and IS2 regions of CAPN3. Here we identified a sibling pair with LGMD2A-type muscular dystrophy caused by a homozygous Ser606Leu (S606L) substitution in the IS2 linker domain. Normal protein levels, unaltered myofibrillar targeting and conserved calcium-induced autocatalytic activity of the mutated protein could be demonstrated in muscle biopsies from one patient. Despite this inconspicuous modification of the IS2 linker between domains III and IV, both patients developed signs and symptoms of the disease within their second decade of life. The unexpected severity of the clinical manifestation points to the high relevance of the calpain 3-specific IS2 segment between domains III and IV. We conclude that the structural motif around the Ser606 residue represents an important functional site that may regulate the transient activation and limited proteolysis of calpain 3.

Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2.

During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date, the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays, colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.

Characterization and Regulation of Lens-specific Calpain Lp82

Eye tissues contain splice variants of muscle-preferred p94 (calpain 3), such as lens-specific Lp82 and Lp85, retina-specific Rt88, and cornea-specific Cn94. The purpose of the present experiment was to analyze the activation and regulation of the best characterized p94 splice variant, Lp82. Recombinant rat Lp82 (rLp82) was expressed using the baculovirus system, purified with Ni-NTA affinity and DEAE-ion exchange chromatographies, and characterized by SDS-PAGE, casein zymography, and immunoblotting. After incubation with calcium, rLp82 autolyzed into two major fragments at approximately 60 and 22 kDa. Sequencing of the autolytic fragments showed loss of three amino acids from the N terminus and cleavage near the IS2 region. Also, Lp82 and calpain 2 were found to hydrolyze each other. Calpastatin inhibited calpain 2 activity, but not Lp82. Homology modeling suggested that the lack of inhibition of Lp82 by calpastatin was due to molecular clashes at the unique AX1 region of Lp82. Lp82 also hydrolyzed calpastatin. These results suggested that Lp82 might regulate other calpain activities and cause hydrolysis of substrates such as crystallins during lens cataract formation.

Identification and characterization of a retina-specific calpain (Rt88) from rat

Purpose. To identify and characterize a newly discovered calpain termed Rt88 from rat retina. Methods. Rt88 in retina under normal physiological conditions was characterized in Sprague-Dawley rats of various ages by competitive RT-PCR, Northern blot analysis, cDNA cloning and sequencing. Recombinant Rt88 was expressed in the baculovirus system and characterized by casein zymography and immunoblotting. Results. Rt88 was sequenced and found to be similar to muscle calpain p94 except for three differences. A different exon 1 (as in lens Lp82 calpain) was present, and exons 15 and 16 in the unique IS2 region of muscle p94 were deleted. Of eleven tissues studied, mRNA for Rt88 was found only in retina where Rt88 increased with maturation and then remained constant. Casein zymography showed that rRt88 was proteolytically active after activation by calcium, but intact rRt88 was rapidly broken due to the presence of the IS1 region in domain II. Conclusions. Rt88 is a retina-specific, calcium activated protease from the calpain superfamily (EC 3.4.22.17) of cysteine proteases. Rt88 is a recently identified member of the AX1 subfamily of calpains showing alternative exon 1 usage. So far, all AX1 subfamily members are from eye. Rt88 may perform specific proteolytic functions during development, normal turnover, or pathological degeneration of retinal proteins.

Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events.

Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.

Lp82 is the Dominant Form of Calpain in Young Mouse Lens

The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC.The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a uniqueN-terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.

719 Expression of interleukin-1 receptor antagonist protein in Alzheimer's Disease, pick's disease and control cases

The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC.The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a uniqueN-terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.

Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle

We previously identified a third type of the calpain large subunit named p94 as a cDNA whose mRNA is expressed exclusively in skeletal muscle at levels approximately 10-fold more abundant than those of the conventional calpain subunit. Rat skeletal muscle fractions were screened by two anti-peptide antibodies raised against two specific sequences in p94, but the p94 protein could not be found. To examine this apparent discrepancy between the amounts of mRNA and protein, wild-type p94 was expressed in COS cells. Although p94 mRNA was expressed normally in COS cells, only very small amounts of the protein and its presumed degradation products were detected by the antibodies described above. A series of COOH-terminal deletion mutants was constructed and expressed in COS cells and L8 cells, a rat myoblast cell line. When IS2, one of the specific regions of p94, was completely eliminated, the truncated p94 proteins were expressed normally, and the amount of the expressed proteins was at least 100-fold higher than with wild-type p94. Moreover, when site-directed mutagenesis was introduced to change the presumed active-site cysteine of p94 to serine or alanine, the mutated p94 proteins were highly expressed like the IS2-deleted mutants. These results indicate the following. 1) The mRNA for p94 is normally transcribed in COS, L8, and muscle cells; 2) the p94 protein becomes active in the cytosol immediately after translation; 3) the p94 protein virtually disappears from cells by autocatalytic degradation; and 4) the p94-specific IS2 region plays an important role in this degradation. In vitro translation experiments support this idea. Furthermore, p94 shows nuclear localization when expressed in COS cells. The physiological function of p94 in muscle is discussed on the basis of the analysis of these transfectants.

DNA Sequence of the mini-insertion IS2–6 and its relation to the sequence of IS2

In polar IS2 abolishes galactose operon expression. Operon activity is restored by a 108 base pair mini-insertion within IS2 called IS2--6. The DNA sequences of the gal operon-IS2 junction, the parental IS2 region undergoing sequence rearrangements and IS2--6 itself are reported. IS2--6 is composed of sequence intervals present in both strands of IS2.

Further mapping of IS2 and IS3 in the lac-purE region of the Escherichia coli K-12 genome: structure of the F-prime ORF203

The sequence organization of the F-prime ORF203 was determined by heteroduplex analysis. This large, type II F-prime (Scaife, 1967) contains lac, proC, and purE genes derived from the W1485 subline of Escherichia coli K-12. The IS3 and IS2 elements previously found in the lac-proC-purE region derived from the 58-161 subline (Hu et al., 1975) are also present in the same locations in the bacterial deoxyribonucleic acid (DNA) from the W1485 subline. Recombination between the IS2 region of F and an IS2 element located between lac and proC on the bacterial DNA apparently led to the formation of the perental Hfr, OR21. IS2 is thus directly repeated, with one copy of each element appearing at each of the two junctions between F and the bacterial sequences on ORF203. The F plasmid is found together with ORF203 in the plasmid DNA, and this probably forms from ORF203 by recombination between the directly repeated IS2 elements. ORF203 appears to have been excised from the Hfr chromosome by recombination between the IS3 sequence alpha3beta3 located counterclockwise of lac and the directly repeated IS3 sequence alpha4beta4 located clockwise of purE.