Expression and characterization of the intact N-terminal domain of streptokinase.
(1/1842)
Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591). The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein. (+info)
Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro.
(2/1842)
Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), is one of at least eight inherited neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein. Here we present two lines of evidence implicating the ubiquitin-proteasome pathway in SCA3/MJD pathogenesis. First, studies of both human disease tissue and in vitro models showed redistribution of the 26S proteasome complex into polyglutamine aggregates. In neurons from SCA3/MJD brain, the proteasome localized to intranuclear inclusions containing the mutant protein, ataxin-3. In transfected cells, the proteasome redistributed into inclusions formed by three expanded polyglutamine proteins: a pathologic ataxin-3 fragment, full-length mutant ataxin-3 and an unrelated GFP-polyglutamine fusion protein. Inclusion formation by the full-length mutant ataxin-3 required nuclear localization of the protein and occurred within specific subnuclear structures recently implicated in the regulation of cell death, promyelocytic leukemia antigen oncogenic domains. In a second set of experiments, inhibitors of the proteasome caused a repeat length-dependent increase in aggregate formation, implying that the proteasome plays a direct role in suppressing polyglutamine aggregation in disease. These results support a central role for protein misfolding in the pathogenesis of SCA3/MJD and suggest that modulating proteasome activity is a potential approach to altering the progression of this and other polyglutamine diseases. (+info)
Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein.
(3/1842)
We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding. (+info)
Amylopectinosis in fetal and neonatal Quarter Horses.
(4/1842)
Three Quarter Horses, a stillborn filly (horse No. 1), a female fetus aborted at approximately 6 months of gestation (horse No. 2), and a 1-month-old colt that had been weak at birth (horse No. 3), had myopathy characterized histologically by large spherical or ovoid inclusions in skeletal and cardiac myofibers. Smaller inclusions were also found in brain and spinal cord and in some cells of all other tissues examined. These inclusions were basophilic, red-purple after staining with periodic acid-Schiff (both before and after digestion with diastase), and moderately dark blue after staining with toluidine blue. The inclusions did not react when stained with Congo red. Staining with iodine ranged from pale blue to black. Their ultrastructural appearance varied from amorphous to somewhat filamentous. On the basis of staining characteristics and diastase resistance, we concluded that these inclusions contained amylopectin. A distinctly different kind of inclusion material was also present in skeletal muscle and tongue of horse Nos. 1 and 3. These inclusions were crystalline with a sharply defined ultrastructural periodicity. The crystals were eosinophilic and very dark blue when stained with toluidine blue but did not stain with iodine. Crystals sometimes occurred freely within the myofibers but more often were encased by deposits of amylopectin. This combination of histologic and ultrastructural features characterizes a previously unreported storage disease in fetal and neonatal Quarter Horses, with findings similar to those of glycogen storage disease type IV. We speculate that a severe inherited loss of glycogen brancher enzyme activity may be responsible for these findings. The relation of amylopectinosis to the death of the foals is unknown. (+info)
Solution properties of the free and DNA-bound Runt domain of AML1.
(5/1842)
The Runt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1 Dm(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain structure or DNA-binding affinity. Purified AML1 Dm(46-185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-specific DNA binding. The NMR spectrum of AML1 Dm(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N-labelled AML1 Dm(46-185) with a 14-bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding. (+info)
Biochemical characterization of the small heat shock protein IbpB from Escherichia coli.
(6/1842)
Escherichia coli IbpB was overexpressed in a strain carrying a deletion in the chromosomal ibp operon and purified by refolding. Under our experimental conditions, IbpB exhibited pronounced size heterogeneity. Basic oligomers, roughly spherical and approximately 15 nm in diameter, interacted to form larger particles in the 100-200-nm range, which themselves associated to yield loose aggregates of micrometer size. IbpB suppressed the thermal aggregation of model proteins in a concentration-dependent manner, and its CD spectrum was consistent with a mostly beta-pleated secondary structure. Incubation at high temperatures led to a partial loss of secondary structure, the progressive exposure of tryptophan residues to the solvent, the dissociation of high molecular mass aggregates into approximately 600-kDa oligomers, and an increase in surface hydrophobicity. Structural changes were reversible between 37 and 55 degrees C, and, up to 55 degrees C, hydrophobic sites were reburied upon cooling. IbpB exhibited a biphasic unfolding trend upon guanidine hydrochloride (GdnHCl) treatment and underwent comparable conformational changes upon melting and during the first GdnHCl-induced transition. However, hydrophobicity decreased with increasing GdnHCl concentrations, suggesting that efficient exposure of structured hydrophobic sites involves denaturant-sensitive structural features. By contrast, IbpB hydrophobicity rose at high NaCl concentrations and increased further at high temperatures. Our results support a model in which temperature-driven conformational changes lead to the reversible exposure of normally shielded binding sites for nonnative proteins and suggest that both hydrophobicity and charge context may determine substrate binding to IbpB. (+info)
The ultrastructure of corneal epithelium after co-cultivation with herpes simplex virus.
(7/1842)
To elucidate the ultrastructural change of corneal epithelium co-cultured with herpes simplex virus (HSV), the corneal epithelium of 3 rabbits was excised and cultivated in culture media. After 7 days, the Kos strain of herpes simplex virus was inoculated in the cultured cornea epithelium until cytopathic effect was occurred. It was fixed in the solution of 3% glutaraldehyde and examined with electronmicroscope. In co-cultured cells, the intercellular spaces had increased and microvilli were seen prominently. The virus particles that initiated the infection by fusing the viral envelope with the plasma membrane were also seen. The nuclear degeneration in an infected cell was prominent. The nuclear membrane was folded markedly, and the chromatin was degraded, condensed and displaced toward the nuclear membrane. Numerous viral particles and inclusion bodies were present in the nuclei. These findings suggest that the infectious process of herpes simplex virus in the human corneal epithelium may occur in a similar way. This result would be helpful in understanding the pathogenesis of herpes simplex epithelial keratitis. (+info)
Angiographic abnormalities in progressive multifocal leukoencephalopathy: an explanation based on neuropathologic findings.
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BACKGROUND AND PURPOSE: Progressive multifocal leukoencephalopathy (PML) is typically occult at angiography and fails to enhance on MR images. After observing angiographic abnormalities characterized by arteriovenous shunting and pathologic parenchymal blush in patients with AIDS-related PML, often in the absence of contrast enhancement on MR images, we hypothesized that there might be distinct changes in the cerebral microvasculature that account for the reduction in vascular transit time (arteriovenous shunting) in the absence of blood-brain barrier dysfunction. METHODS: The imaging studies and neuropathologic specimens of six patients with biopsy-proved PML were reviewed retrospectively. In all patients contrast-enhanced MR imaging and CT, followed by cerebral angiography, were performed before stereotactically directed biopsy. The angiograms were evaluated for the presence of vascular displacement, pathologic parenchymal blush, arteriovenous shunting, and neovascularity. The CT and MR studies were reviewed for the presence of enhancement of the PML lesions. Biopsy specimens were examined for the presence of necrosis, perivascular inflammation, and neovascularity. RESULTS: All patients had oligodendrocytic intranuclear inclusions diagnostic of PML, together with perivascular inflammation and neovascularity to a varying extent; no other neuropathologic processes were identified. Angiographic abnormalities, characterized by a pathologic parenchymal blush and arteriovenous shunting, were identified in four of the six patients. In only one of these cases, however, was abnormal enhancement identified on cross-sectional imaging studies (MR and CT), and this patient had florid perivascular inflammatory infiltrates histologically. CONCLUSION: The pathologic parenchymal blush and arteriovenous shunting seen angiographically in some patients with PML reflect small-vessel proliferation and perivascular inflammatory changes incited by the presence of the JC virus in infected oligodendrocytes. (+info)