Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.
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PMID:Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. 134 95

Brain, spleen, and selected lymph nodes from sheep with clinical signs of scrapie were analyzed for presence of proteinase K-resistant protein (PrP-res). Diagnosis of scrapie on the basis of detection of PrP-res was compared with diagnosis on the basis of histologic evaluation of the brain from clinically affected or exposed sheep. Proteinase K-resistant protein was found in every brain that was histologically positive for scrapie, and in addition, was found in the brain of several clinically positive sheep that were not diagnosed as scrapie-positive by histologic evaluation. Proteinase K-resistant protein was also found in 87% of the spleens and lymph nodes from sheep that had PrP-res detected in brain homogenates. Therefore, analysis of sheep brain, spleen, or lymph nodes for PrP-res provided a diagnostic approach that was superior to histologic examination alone for detection of naturally scrapie agent-infected sheep.
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PMID:Diagnostic implications of detection of proteinase K-resistant protein in spleen, lymph nodes, and brain of sheep. 135 64

A 19-month-old greater kudu (Tragelaphus strepsiceros), whose dam had died 15 months earlier with spongiform encephalopathy, required euthanasia after developing severe ataxia and depression with an apparently sudden onset. No macroscopic abnormalities were detected on post mortem examination but a scrapie-like spongiform encephalomyelopathy was apparent on histopathological examination of brain and segments of spinal cord. Negative stain electron microscopy of proteinase K-treated detergent extracts of tissue from the brain stem revealed the presence of scrapie associated fibrils, and a 25 to 28 kDa band comparable with that identified as abnormal PrP (prion protein) from the brains of domestic cattle with spongiform encephalopathy was detected using rabbit antiserum raised against mouse PrP. The animal was born nine months after the statutory ban on the inclusion of ruminant-derived protein in ruminant feeds and, as no other possible sources of the disease were apparent, it appears likely that the infection was acquired from the dam.
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PMID:Scrapie-like encephalopathy in a greater kudu (Tragelaphus strepsiceros) which had not been fed ruminant-derived protein. 160 83

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.
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PMID:Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. 167 78

Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a protease-resistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapie-associated modification of PrP, we have studied the processing and sedimentation properties of protease-resistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.
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PMID:N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. 168 7

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.
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PMID:Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. 196 66

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.
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PMID:Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. 197 60

The scrapie and cellular isoforms of the prion protein (PrPSc and PrPC) differ strikingly in a number of their biochemical and metabolic properties. The structural features underlying these differences are unknown, but they are thought to result from a posttranslational process. Both PrP isoforms contain complex type oligosaccharides, raising the possibility that differences in the asparagine-linked glycosylation account for the properties that distinguish PrPC and PrPSc. ScN2a and ScHaB cells in culture produce several PrP molecules with relative molecular masses of 26-35 kDa and proteinase K-resistant cores of 19-29 kDa. When the cells were treated with tunicamycin, this heterogeneity was eliminated and a single PrP species of 26 kDa was observed. Several hours after its synthesis, a fraction of this protein became insoluble in detergents and acquired a proteinase K-resistant core, thus displaying two of the biochemical hallmarks of PrPSc. Synthesis in the presence of tunicamycin restricted the proteinase K-resistant cores of PrP to a single species of 19 kDa. No proteinase K-resistant PrP was found in uninfected cells. Expression of a mutated PrP gene lacking both asparagine-linked glycosylation sites in ScN2a cells resulted in the synthesis of 19-kDa proteinase K-resistant PrP molecules. We conclude that asparagine-linked glycosylation is not essential for the synthesis of proteinase K-resistant PrP and that structural differences unrelated to asparagine-linked oligosaccharides must exist between PrPC and PrPSc. Whether unglycosylated PrPSc molecules are associated with scrapie prion infectivity remains to be established.
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PMID:Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation. 197 22

Scrapie-associated fibrils (SAF) are a ubiquitous pathological feature of brains affected by scrapie and the other scrapie-like agents. They are composed of PrP, a heterogeneous glycoprotein which is also present in normal brain but not as SAF. The PrP protein associated with SAF is partially resistant to proteinase K, whereas the soluble form is not. It has been proposed that SAF do not exist as such in vivo, but rather self-assemble from subunit structures liberated from membranes by detergent extraction during purification. We have purified SAF by a method that does not employ proteinase K. We show that the PrP protein from infected but not uninfected brain is partially resistant to protease digestion before and after detergent extraction. Likewise, SAF can be sheared by sonication before or after detergent extraction. In addition, SAF from mice infected with different strains of scrapie have different sedimentation properties. Since SAF-dependent properties exist before detergent extraction, then so must SAF. They are therefore not a detergent-induced artefact but most probably assemble in vivo.
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PMID:Structural and biochemical evidence that scrapie-associated fibrils assemble in vivo. 256 38

A number of studies have indicated that an endogenous brain protein, PrP, is associated with transmissible agents causing spongiform encephalopathies such as scrapie, kuru, and Creutzfeldt-Jakob disease. It has been proposed that PrP derived from scrapie brain is the scrapie agent itself. To test directly whether the PrP mRNA in scrapie brain tissue can encode the scrapie agent, we expressed PrP cDNA cloned from scrapie-infected mouse brain in vitro. The expressed PrP did not transmit scrapie to susceptible mice. Thus either PrP is not the scrapie agent, or the expressed PrP requires additional modification to be infectious. The normal function of PrP is unknown, however, comparison of the amino acid sequences of PrP from mouse, hamster, and human revealed that many structural features of potential functional significance have been conserved during evolution. To learn about normal PrP and whether it is altered by scrapie infection in vitro, we have performed studies of PrP biosynthesis in normal and scrapie-infected mouse neuroblastoma tissue culture cells. The major PrP species were glycoproteins anchored at the cell surface by covalent linkage to phosphatidylinositol. No scrapie-associated modifications of PrP biosynthesis were observed, and, none of the metabolically labeled PrP observed in either scrapie-infected normal cells was resistant to proteinase K.
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PMID:Comparative sequence analysis, in vitro expression and biosynthesis of mouse PrP. 257 73


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