Adriana Ramos

PK-sensitive PrPSc Is Infectious and Shares Basic Structural Features with PK-resistant PrPSc

One of the main characteristics of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrPSc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrPSc that is sensitive to PK hydrolysis (sPrPSc). 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 sPrPSc versus the PK-resistant (rPrPSc) fractions of PrPSc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrPSc and rPrPSc 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 sPrPSc to the rPrPSc forms of PrPSc. Although the sPrPSc and rPrPSc 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.

Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus

We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride (Ac(2)O), which specifically target accessible tyrosine and lysine residues, respectively, to modify recombinant Syrian hamster PrP(90-231) [rSHaPrP(90-231)] and SHaPrP 27-30, the proteinase K-resistant core of PrP(Sc) isolated from brain of scrapie-infected Syrian hamsters. Our aim was to find locations of conformational change. Modified proteins were subjected to in-gel proteolytic digestion with trypsin or chymotrypsin and subsequent analysis by mass spectrometry (MALDI-TOF). Several differences in chemical reactivity were observed. With TNM, the most conspicuous reactivity difference seen involves peptide E(221)-R(229) (containing Y(225) and Y(226)), which in rSHaPrP(90-231) was much more extensively modified than in SHaPrP 27-30; peptide H(111)-R(136), containing Y(128), was also more modified in rSHaPrP(90-231). Conversely, peptides Y(149)-R(151), Y(157)-R(164), and R(151)-Y(162) suffered more extensive modification in SHaPrP 27-30. Acetic anhydride modified very extensively peptide G(90)-K(106), containing K(101), K(104), K(106), and the amino terminus, in both rSHaPrP(90-231) and SHaPrP 27-30. These results suggest that (1) SHaPrP 27-30 exhibits important conformational differences in the C-terminal region with respect to rSHaPrP(90-231), resulting in the loss of solvent accessibility of Y(225) and Y(226), very solvent-exposed in the latter conformation; because other results suggest preservation of the two C-terminal helices, this might mean that these are tightly packed in SHaPrP 27-30. (2) On the other hand, tyrosines contained in the stretch spanning approximately Y(149)-R(164) are more accessible in SHaPrP 27-30, suggesting rearrangements in α-helix H1 and the short β-sheet of rSHaPrP(90-231). (3) The amino-terminal region of SHaPrP 27-30 is very accessible. These data should help in the validation and construction of structural models of PrP(Sc).

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

Structural Organization of Mammalian Prions as Probed by Limited Proteolysis

Elucidation of the structure of PrPSc 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 (PrPSc). 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 PrPSc 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 PrPSc 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 PrPSc. These results reinforce the hypothesis that the structure of PrPSc consists of a series of highly PK-resistant β-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrPSc is highly resistant to PK and therefore perhaps also contains β-sheet secondary structure.

A new seipin-associated neurodegenerative syndrome

Background Seipin/BSCL2 mutations can cause type 2 congenital generalised lipodystrophy (BSCL) or dominant motor neurone diseases. Type 2 BSCL is frequently associated with some degree of intellectual impairment, but not to fatal neurodegeneration. In order to unveil the aetiology and pathogenetic mechanisms of a new neurodegenerative syndrome associated with a novel BSCL2 mutation, six children, four of them showing the BSCL features, were studied. Methods Mutational and splicing analyses of BSCL2 were performed. The brain of two of these children was examined postmortem. Relative expression of BSCL2 transcripts was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in different tissues of the index case and controls. Overexpressed mutated seipin in HeLa cells was analysed by immunofluorescence and western blotting. Results Two patients carried a novel homozygous c.985C>T mutation, which appeared in the other four patients in compound heterozygosity. Splicing analysis showed that the c.985C>T mutation causes an aberrant splicing site leading to skipping of exon 7. Expression of exon 7-skipping transcripts was very high with respect to that of the non-skipped transcripts in all the analysed tissues of the index case. Neuropathological studies showed severe neurone loss, astrogliosis and intranuclear ubiquitin(+) aggregates in neurones from multiple cortical regions and in the caudate nucleus. Conclusions Our results suggest that exon 7 skipping in the BSCL2 gene due to the c.985C>T mutation is responsible for a novel early onset, fatal neurodegenerative syndrome involving cerebral cortex and basal ganglia.

SAXS structural study of PrPSc reveals ~11 nm diameter of basic double intertwined fibers

A sample of purified Syrian hamster PrP27–30 prion fibers was analyzed by synchrotron small-angle X-ray scattering (SAXS). The SAXS pattern obtained was fitted to a model based on infinitely long cylinders with a log-normal intensity distribution, a hard-sphere structure factor and a general Porod term for larger aggregates. The diameter calculated for the cylinders determined from the fit was 11.0 ± 0.2 nm. This measurement offers an estimation of the diameter of PrPSc fibers in suspension, i.e., free of errors derived from estimations based on 2D projections in transmission electron microscopy images, subjected to further possible distortions from the negative stain. This diameter, which corresponds to a maximum diameter of approximately 5.5 nm for each of the two intertwined protofilaments making up the fibers, rules out the possibility that PrPSc conforms to a stack of in-register, single-rung flat PrPSc monomers; rather, PrPSc subunits must necessarily coil, most likely several times, into themselves.

Role of Apoptosis Signal-regulating Kinase 1 (ASK1) as an Activator of the GAPDH-Siah1 Stress-Signaling Cascade

Background: Apoptosis signal-regulating kinase 1 (ASK1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and seven in absentia homolog 1 (Siah1) are molecules associated with stress-signaling cascades. Results: Identification of Siah1 as a substrate of ASK1 for activation of the GAPDH-Siah1 signaling cascade. Conclusion: ASK1 triggers the GAPDH-Siah1 stress-signaling cascade. Significance: This study provides insight into crosstalk among cell stress-signaling cascades. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays roles in both energy maintenance, and stress signaling by forming a protein complex with seven in absentia homolog 1 (Siah1). Mechanisms to coordinate its glycolytic and stress cascades are likely to be very important for survival and homeostatic control of any living organism. Here we report that apoptosis signal-regulating kinase 1 (ASK1), a representative stress kinase, interacts with both GAPDH and Siah1 and is likely able to phosphorylate Siah1 at specific amino acid residues (Thr-70/Thr-74 and Thr-235/Thr-239). Phosphorylation of Siah1 by ASK1 triggers GAPDH-Siah1 stress signaling and activates a key downstream target, p300 acetyltransferase in the nucleus. This novel mechanism, together with the established S-nitrosylation/oxidation of GAPDH at Cys-150, provides evidence of how the stress signaling involving GAPDH is finely regulated. In addition, the present results imply crosstalk between the ASK1 and GAPDH-Siah1 stress cascades.

Neuropeptide precursor VGF is genetically associated with social anhedonia and underrepresented in the brain of major mental illness: its downregulation by DISC1

In a large Scottish pedigree, disruption of the gene coding for DISC1 clearly segregates with major depression, schizophrenia and related mental conditions. Thus, study of DISC1 may provide a clue to understand the biology of major mental illness. A neuropeptide precursor VGF has potent antidepressant effects and has been reportedly associated with bipolar disorder. Here we show that DISC1 knockdown leads to a reduction of VGF, in neurons. VGF is also downregulated in the cortices from sporadic cases with major mental disease. A positive correlation of VGF single-nucleotide polymorphisms (SNPs) with social anhedonia was also observed. We now propose that VGF participates in a common pathophysiology of major mental disease.

Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease

Celias Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers.