PK-sensitive PrP is infectious and shares basic structural features with PK-resistant PrP.

One of the main characteristics of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP(Sc) following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP(Sc) that is sensitive to PK hydrolysis (sPrP(Sc)). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP(Sc) versus the PK-resistant (rPrP(Sc)) fractions of PrP(Sc) and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP(Sc) and rPrP(Sc) fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP(Sc) to the rPrP(Sc) forms of PrP(Sc). Although the sPrP(Sc) and rPrP(Sc) fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.

Diagnosis and treatment of hepatocellular carcinoma. Update of the consensus document of the AEEH, AEC, SEOM, SERAM, SERVEI, and SETH. / Diagnóstico y tratamiento del carcinoma hepatocelular. Actualización del documento de consenso de la AEEH, AEC, SEOM, SERAM, SERVEI y SETH.

Hepatocellular carcinoma (HCC) is the most common primary liver neoplasm and one of the most common causes of death in patients with cirrhosis of the liver. In parallel, with recognition of the clinical relevance of this cancer, major new developments have recently appeared in its diagnosis, prognostic assessment and in particular, in its treatment. Therefore, the Spanish Association for the Study of the Liver (AEEH) has driven the need to update the clinical practice guidelines, once again inviting all the societies involved in the diagnosis and treatment of this disease to participate in the drafting and approval of the document: Spanish Society for Liver Transplantation (SETH), Spanish Society of Diagnostic Radiology (SERAM), Spanish Society of Vascular and Interventional Radiology (SERVEI), Spanish Association of Surgeons (AEC) and Spanish Society of Medical Oncology (SEOM). The clinical practice guidelines published in 2016 and accepted as National Health System Clinical Practice Guidelines were taken as the reference documents, incorporating the most important recent advances. The scientific evidence and the strength of the recommendation is based on the GRADE system.

Fibrillar prion peptide PrP(106-126) treatment induces Dab1 phosphorylation and impairs APP processing and Abeta production in cortical neurons.

Alzheimer's disease and prion diseases (e.g., Creutzfeldt-Jakob disease) display profound neural lesions associated with aberrant protein processing and extracellular amyloid deposits. However, the intracellular events in prion diseases and their relation with the processing of the amyloid precursor protein (APP) and beta-amyloid generation are unknown. The adaptor protein Dab1 may regulate intracellular trafficking and secretase-mediated proteolysis in APP processing. However, a putative relationship between prion diseases and Dab1/APP interactions is lacking. Thus, we examined, in inoculated animals, whether Dab1 and APP processing are targets of the intracellular events triggered by extracellular exposure to PrP(106-126) peptide. Our in vitro results indicate that PrP(106-126) peptide induces tyrosine phosphorylation of Dab1 by activated members of the Src family of tyrosine kinases (SFK), which implies further Dab1 degradation. We also corroborate these results in Dab1 protein levels in prion-inoculated hamsters. Finally, we show that fibrillar prion peptides have a dual effect on APP processing and beta-amyloid production. First, they block APP trafficking at the cell membrane, thus decreasing beta-amyloid production. In parallel, they reduce Dab1 levels, which also alter APP processing. Lastly, neuronal cultures from Dab1-deficient mice showed severe impairment of APP processing with reduced sAPP secretion and A beta production after prion peptide incubation. Taken together, these data indicate a link between intracellular events induced by exposure to extracellular fibrillar peptide or PrP(res), and APP processing and implicate Dab1 in this link.

Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease.

The basic molecular underpinnings of the pathological changes that unfold in prion disease remain elusive. A key role of increased oxidative stress has been hypothesized. Given the transient nature of most intermediate molecules implicated, increased oxidative stress is better assessed by quantitating the damage it causes to macromolecules. We used mass spectrometry-based methods to measure specific products of protein oxidation, glycoxidation, and lipoxidation in brains from patients suffering from Creutzfeldt-Jakob disease and Syrian hamsters affected by scrapie. In both cases, increased amounts of glutamic and aminoadipic semialdehydes, products of metal-catalyzed oxidation, malondialdehydelysine (a product of lipoxidation), N-epsilon-carboxyethyllysine (a product of glycoxidation), and N-epsilon-carboxymethyllysine (generated by lipoxidation and glycoxidation) were measured. PrP(Sc), the infectious isoform of the prion protein that accumulates in prion disease, was itself shown to be a target of increased oxidative modification. These changes were accompanied by alterations in fatty acid composition and increased phosphorylation of ERK(1/2) and p38, protein kinases known to respond to increased flows of ROS. These data support an important role of oxidative damage in the pathology of prion disease.

Isolation and characterization of a proteinase K-sensitive PrPSc fraction.

Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.

Structural organization of mammalian prions as probed by limited proteolysis.

Elucidation of the structure of PrP(Sc) continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP(Sc)). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrP(Sc) from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP(Sc) samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP(Sc). These results reinforce the hypothesis that the structure of PrP(Sc) consists of a series of highly PK-resistant ß-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP(Sc) is highly resistant to PK and therefore perhaps also contains ß-sheet secondary structure.

Scrapie prion protein structural constraints obtained by limited proteolysis and mass spectrometry.

Elucidation of the structure of scrapie prion protein (PrP(Sc)), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP(Sc). Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in beta-strands. We treated PrP(Sc) isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP(Sc) was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrP(Sc) confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP(Sc) are susceptible to PK. Our results indicate that besides the "classic" amino-terminal PK cleavage points, PrP(Sc) contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short beta-sheet stretches connected by loops and turns.