SMYD1b is co-regulated with other myosin chaperones HSP90a1 and UNC45b.

<p>In-situ hybridization staining of wild type and <i>still heart</i> embryos shows that <i>hsp90a1</i> expression is normal in <i>still heart</i> mutants, when compared to wild type embryos at 19hpf (A&B). However, the expression of <i>hsp90a1</i> and <i>unc45b</i> dramatically increases in <i>still heart</i> mutants at 24hpf (C-F & K) and 48hpf throughout the somites (G-J & M). Additionally, <i>smyd1b</i> expression increases significantly when UNC45b is absent in <i>steif</i> mutants (N), supporting co-regulation of these three genes. (O) A time course of <i>smyd1b</i> expression during muscle formation reveals that <i>smyd1b</i> is expressed early at 10hpf when <i>unc45b</i> is expressed and increases in its expression as muscle development progresses. Due to the rapid development of <i>unc45b</i> staining in the somites of the embryo in panel J, the head while present, has no background staining and is not clear in this focal plane. (qPCR: n = 3, 30 embryos each time/phenotype. Error bars are standard deviation.).</p

<i>Still Heart</i> Encodes a Structural HMT, SMYD1b, with Chaperone-Like Function during Fast Muscle Sarcomere Assembly

<div><p>The vertebrate sarcomere is a complex and highly organized contractile structure whose assembly and function requires the coordination of hundreds of proteins. Proteins require proper folding and incorporation into the sarcomere by assembly factors, and they must also be maintained and replaced due to the constant physical stress of muscle contraction. Zebrafish mutants affecting muscle assembly and maintenance have proven to be an ideal tool for identification and analysis of factors necessary for these processes. The <i>still heart</i> mutant was identified due to motility defects and a nonfunctional heart. The cognate gene for the mutant was shown to be <i>smyd1b</i> and the <i>still heart</i> mutation results in an early nonsense codon. SMYD1 mutants show a lack of heart looping and chamber definition due to a lack of expression of heart morphogenesis factors <i>gata4</i>, <i>gata5</i> and <i>hand2</i>. On a cellular level, fast muscle fibers in homozygous mutants do not form mature sarcomeres due to the lack of fast muscle myosin incorporation by SMYD1b when sarcomeres are first being assembled (19hpf), supporting SMYD1b as an assembly protein during sarcomere formation.</p></div

<i>Still heart</i>, a <i>smyd1b</i> mutant, has defects in heart and fast skeletal muscle tissue.

<p>A lateral view of 48hpf wild type (A) and <i>still heart</i> (<i>sth</i>) mutants (B), which have pericardial edema, small eyes, malformed head and reduced motility. Black arrowheads highlight the pericardial edema in sth mutants and the absence of edema in wild type. White arrowheads indicate blood pooling in the mutant and the absence of pooling in wild type. (C&D) <i>Sth</i> mutant hearts are underdeveloped and do not beat. (E-G) Examination of lateral myofibers at 5dpf under DIC microscopy revealed striations, indicative of fully formed sarcomeres, are visible in the myofibers of wild type muscle (E, black arrowheads), while absent in the fast muscle of <i>still heart</i> mutants (F); striations are present in <i>sth</i> slow muscle (G, black arrowhead) but are disturbed by nuclei and fluid-filled spaces (G, white arrowhead). Sequencing of <i>smyd1b</i> cDNA from wild type embryos (H) and <i>sth</i> mutant embryos (I) revealed a 9 nucleotide insertion between exon 1 and 2 in the <i>smyd1b</i> mRNA, creating an in-frame stop codon (I, underlined sequence). The insertion is the first 9 nucleotides of intron one as sequenced from wild type smyd1b genomic sequence (J). This is a result of a transition mutation in the splice donor site of intron 1 (I, green letter in sequence, J, outlined letter in sequence). This results in a premature truncation of the SMYD1b protein after exon 1 (K).</p

SMYD1b is required for fast myosin incorporation during sarcomere assembly.

<p>At 48hpf, fast myosin (F310—green) and actin (phalloidin—red) staining is visible in the premyofibrils in wt zebrafish tails (A&B), while absent from the premyofibrils in <i>sth</i> fast muscle tissue (C&D). Slow muscle (F59) develops normally in both wild type and <i>still heart</i> zebrafish at 48hpf (E&F, G&H). At 19hpf, in wild type embryos, fast myosin (F310) is beginning to be incorporated into the maturing myofibril (I) and overlaps (white arrowheads) with the developing actin (phalloidin) (J) fibers in trunk muscle (K, merge, K’ inset, white arrowhead). Fast myosin is not incorporated into the maturing premyofibril (L), although actin fibers are still present (M&N, N’ inset).</p

A novel Gerstmann-Sträussler-Scheinker disease mutation defines a precursor for amyloidogenic 8 kDa PrP fragments and reveals N-terminal structural changes shared by other GSS alleles

<div><p>To explore pathogenesis in a young Gerstmann-Sträussler-Scheinker Disease (GSS) patient, the corresponding mutation, an eight-residue duplication in the hydrophobic region (HR), was inserted into the wild type mouse PrP gene. Transgenic (Tg) mouse lines expressing this mutation (Tg.HRdup) developed spontaneous neurologic syndromes and brain extracts hastened disease in low-expressor Tg.HRdup mice, suggesting <i>de novo</i> formation of prions. While Tg.HRdup mice exhibited spongiform change, PrP aggregates and the anticipated GSS hallmark of a proteinase K (PK)-resistant 8 kDa fragment deriving from the center of PrP, the LGGLGGYV insertion also imparted alterations in PrP's unstructured N-terminus, resulting in a 16 kDa species following thermolysin exposure. This species comprises a plausible precursor to the 8 kDa PK-resistant fragment and its detection in adolescent Tg.HRdup mice suggests that an early start to accumulation could account for early disease of the index case. A 16 kDa thermolysin-resistant signature was also found in GSS patients with P102L, A117V, H187R and F198S alleles and has coordinates similar to GSS stop codon mutations. Our data suggest a novel shared pathway of GSS pathogenesis that is fundamentally distinct from that producing structural alterations in the C-terminus of PrP, as observed in other prion diseases such as Creutzfeldt-Jakob Disease and scrapie.</p></div

Ontogeny and properties of abnormal PrP species in Tg.HRdup mice.

<p>A) In vitro proteolysis products of HRDup and WT PrP derived in young animals. The hydrophobic region (HR) is presented in orange, helices in dark blue, beta strands in red and all other residues indicated by mid-blue shading. The approximate location of the epitopes of the anti-PrP antibodies used to map the boundaries of the protease resistant domains are indicated. In the presence of thermolysin (TL) and proteinase K (PK) WT PrP^C is completely degraded (indicated by dotted outline). In contrast, the disposition of HRdup PrP is different, with conformational effects of the 8-residue insert (yellow) insert spreading both in an N-terminal direction (horizontal black arrow) and in a C-terminal direction (horizontal grey arrow); these changes in conformation are indicated by the mid-blue shading of PrP^C being replaced by wavy lines. While being completely degraded by PK (like PrP^C) HRdup can adopt a conformation whereby the N-terminal region is TL-resistant and hence only the C-terminal portion of the molecule is degraded by this protease. From gel analyses the cleavage sites may be heterogeneous ("ragged termini"); these termini have not been mapped in detail but are shown in a provisional manner by small vertical arrows. The action of C1 protease (large vertical arrow) immediately N-terminal to the HR would preclude the formation of 16 or 8 kDa protease-resistant species. B) Proposed evolution of protease resistant PrP species from HRdup PrP. For simplicity, ragged termini have been omitted from this schematic. The aging process is shown on the vertical axis. The signature 16 kDa thermolysin species of the HRdup PrP is already present in young mice, exhibits a slow accumulation with aging but remains PK sensitive. A similar species may accumulate spontaneously from endogenous protease action without the need for in vitro thermolysin digestion (asterisk). At a later stage in the disease course the 16kDa species are hypothesized to undergo a different conformational change (indicated by dark blue wavy fill) such that its C-terminal region (i.e. the central region of PrP) acquires resistance to endogenous proteases and accumulates; it also has the property of being resistant to PK digestion performed in vitro and yields the GSS signature 8 kDa PK-resistant fragment. The 8 kDa species can be amplified by templated refolding as shown in transmission experiments. Some full-length protease-resistant PrP may also be present at disease end-stage (e.g., <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006826#ppat.1006826.g007" target="_blank">Fig 7C</a>, lane 6) but this is neither the predominant species nor consistently present and hence is not represented here. The presence of 16 kDa TL-resistant species in a number of GSS cases containing 7–8 kDa PK-resistant species (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006826#ppat.1006826.g006" target="_blank">Fig 6E</a>) suggest that this scheme may be generally applicable.</p

HRdup PrP causes GSS in transgenic mice.

<p>A) Line diagram of PrP demonstrating the site of the 8 amino acid insertion (yellow box) in the center of PrP. The hydrophobic region, "HR", is shown in orange, β-strands (strand 1 and strand 2, "S1" and "S2") are in red and helix A in dark blue. The presence of valine at res. 128 is indicated. B) Kaplan-Meier survival plot of the transgenic lines expressing HRdup PrP. Tg.HRdup-32, 166 ± 39 days (black, SD; n = 9); Tg.HRdup-26, 410 ± 40 days (red, SD; n = 23) and Tg.HRdup-10, 662 ± 76 days (blue, SD; n = 16). Panels C & D, Pathological features of the index GSS patient. C) Focal neocortical moderate spongiosis (middle frontal gyrus), not preferentially associated with MCP. H&E staining, scale bar = 50 μm. D) Low power views of the cortex in H&E, PAS and 12F10 antibody stain (I-III) and a higher power view (IV-VI) with size bars of 500 μm and 25 μm, respectively. E) Pathological features of Tg animals with spontaneous disease. Photomicrographs of sagittal brain sections of Tg.HRdup-26 mice at terminal stage of disease (panels I-XII). Immunostaining for PrP was performed after treatment of the slices with formic acid while slices for examination with H&E (third column) or GFAP (fourth column) were left untreated. The CA1 region of the hippocampus is shown. Inset in panel II shows a PAS-positive plaque in the cortex. Scale bar = 25 μm. F) High power view of cortical (I and II) and cerebellar (III and IV) PrP deposits in Tg.HRdup mice stained with two antibodies, as indicated. Scale bar = 50 μm.</p

NMR spectra and beta-sheet signatures of recombinant proteins.

<p>A) Representative urea titration of one (Y162) of five assayed residues positioned in PrP's globular domain, as assessed for three PrP constructs (as indicated). B) Half maximal urea concentrations of each of the five residues with allelic origin color code as per panel A and presented with residues in numerical order. No pair-wise comparisons between the same residue measured in the three alleles reached significance. C) Position of beta strand S1 in WT PrP (top line) and a potential, additional beta strand, both indicated by S, that might arise as a consequence of the tandem duplication encompassing the residues YVLG. Lower panels; Part of the 2D ¹H-¹H NOESY spectra of M128V (D) and HRdup (E) where NOEs due to the presence of β-sheet are displayed.</p

Expression level, disease onset and signature PrP fragments in transgenic mice.

<p>Expression level, disease onset and signature PrP fragments in transgenic mice.</p

Molecular dynamics assessment of HRdup PrP.

<p>A) Representative conformations from 20ns molecular dynamics runs for M128V and HRdup PrP. α-helices are colored with magenta, β-strands with yellow, turns with cyan, random coils are white and the insert (for HRdup) is red. For the alignment, regions in HRdup that show differences with respect to M128V are depicted in green, and the insert is indicated in red. B) Differences in weighted average per-residue SASA for HRdup versus M128V models were averaged over two trajectories per allele (positive or negative differences indicate, respectively, greater or lesser solvent exposure in HRdup in comparison with M128V, as listed in the last column of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006826#ppat.1006826.s018" target="_blank">S5 Table</a>). Positions of helices and beta strands are shaded in grey (see also panel C). C) Main chain flexibility profiles in the PrP systems. For M128V, the flexibility profile is shown with red and black lines, and for systems with insert the profiles are shown with yellow and blue lines. The insert area is marked with vertical dashed lines. The main secondary structure elements in PrP are shaded in grey are indicated at the top of the plots with purple lines for α-helices and yellow lines for β-strands. Panels D) and E): The strongest correlations of non-consecutive residues from ECD pair correlation maps of the four PrP systems, as listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006826#ppat.1006826.s018" target="_blank">S5 Table</a>. D) Data for M128V-I (red dots) and M128V-II (black dots); E) Data for HRdup-I (blue crosses) and HRdup-II (yellow crosses). The regions of strongest correlations S1-S2, S2-H2, S2-H3, and H2-H3 are indicated. Correlations S1-S2 determined from 2D NOESY experiments are shown with magenta crosses in D and E.</p