Satoshi Takahashi

3D structure of amyloid protofilaments of β2-microglobulin fragment probed by solid-state NMR

Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser20-Lys41) of β2-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly 13C,15N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a β-strand–loop–β-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of β2-microglobulin protofilament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR.

Serum Amyloid A3 Binds MD-2 To Activate p38 and NF-κB Pathways in a MyD88-Dependent Manner

Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20–86) (20–86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20–86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20–86) or SAA3 (43–57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 μM, respectively. FLAG-tagged SAA3 tightly bound to protein A–tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20–86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-β1. An i.v. injection of SAA3 (43–57) induced the lung recruitment of CD11b+Gr-1+ cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.

Effects of soil erosion and anoxic-euxinic ocean in the Permian-Triassic marine crisis.

The largest mass extinction of biota in the Earth’s history occurred during the Permian–Triassic transition and included two extinctions, one each at the latest Permian (first phase) and earliest Triassic (second phase). High seawater temperature in the surface water accompanied by euxinic deep-intermediate water, intrusion of the euxinic water to the surface water, a decrease in pH, and hypercapnia have been proposed as direct causes of the marine crisis. For the first-phase extinction, we here add a causal mechanism beginning from massive soil and rock erosion and leading to algal blooms, release of toxic components, asphyxiation, and oxygen-depleted nearshore bottom water that created environmental stress for nearshore marine animals. For the second-phase extinction, we show that a soil and rock erosion/algal bloom event did not occur, but culmination of anoxia–euxinia in intermediate waters did occur, spanning the second-phase extinction. We investigated sedimentary organic molecules, and the results indicated a peak of a massive soil erosion proxy followed by peaks of marine productivity proxy. Anoxic proxies of surface sediments and water occurred in the shallow nearshore sea at the eastern and western margins of the Paleotethys at the first-phase extinction horizon, but not at the second-phase extinction horizon. Our reconstruction of ocean redox structure at low latitudes indicates that a gradual increase in temperature spanning the two extinctions could have induced a gradual change from a well-mixed oxic to a stratified euxinic ocean beginning immediately prior to the first-phase extinction, followed by culmination of anoxia in nearshore surface waters and of anoxia and euxinia in the shallow-intermediate waters at the second-phase extinction over a period of approximately one million years or more. Enhanced global warming, ocean acidification, and hypercapnia could have caused the second-phase extinction approximately 60 kyr after the first-phase extinction. The causes of the first-phase extinction were not only those environmental stresses but also environmental stresses caused by the soil and rock erosion/algal bloom event.

Hypothesis: structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and the long-range distance distribution

We propose a hypothesis that explains two apparently contradicting observations for the heterogeneity of the unfolded proteins. First, the line confocal method of the single-molecule Förster resonance energy transfer (sm-FRET) spectroscopy revealed that the unfolded proteins possess broad peaks in the FRET efficiency plot, implying the significant heterogeneity that lasts longer than milliseconds. Second, the fluorescence correlation method demonstrated that the unfolded proteins fluctuate in the time scale shorter than 100xa0ns. To formulate the hypothesis, we first summarize the recent consensus for the structure and dynamics of the unfolded proteins. We next discuss the conventional method of the sm-FRET spectroscopy and its limitations for the analysis of the unfolded proteins, followed by the advantages of the line confocal method that revealed the heterogeneity. Finally, we propose that the structural heterogeneity formed by the local clustering of hydrophobic residues modulates the distribution of the long-range distance between the labeled chromophores, resulting in the broadening of the peak in the FRET efficiency plot. A clustering of hydrophobic residues around the chromophore might further contribute to the broadening. The proposed clusters are important for the understanding of protein folding mechanism.

Triple-negative and HER2 positive ductal carcinoma in situ of the breast: characteristics, behavior, and biomarker profile

We compared the characteristics, clinical behavior, and biomarker profile between HER2 positive (HER2+) and triple-negative (TN) ductal carcinoma in situ (DCIS) which are considered more aggressive than other DCIS subtypes. In addition, we explored the impact of these features on its potential of progression to invasive breast carcinomas. Cases of DCIS diagnosed at the Department of Pathology, Singapore General Hospital from 1994 to 2010 were identified. TN and HER2+ DCIS cases formed the study cohort. Immunohistochemistry (IHC) was performed for ER, PR, HER2, CK14, EGFR, and p53. Comparisons of clinicopathological features, IHC results, and clinical outcomes were performed between the two groups. We evaluated 145 HER2+ and 85 TN DCIS cases. HER2 positive DCIS had significantly higher nuclear grade (pu2009<u20090.001) and more frequent necrosis (pu2009<u20090.001) than TN DCIS. HER2 positive DCIS also harbored significantly higher rates of nuclear p53 immunoreactivity (pu2009=u20090.002) than TN DCIS. Younger patients (ageu2009<u200940) with HER2+ and TN DCIS demonstrated statistically significant worse invasive DFS than older women (pu2009<u20090.001). Multivariate cox regression analysis (HR 15.08, 95% CI 12.79–81.45, pu2009=u20090.002) also confirmed these findings. In addition, younger patients (ageu2009<u200940) with HER2+ DCIS experienced significantly poorer prognosis when p53 was also positive (pu2009=u20090.033). HER2+ DCIS had more aggressive pathological characteristics compared to TN DCIS; accumulation of mutant p53 could possibly be contributory. Age was an independent predictor of aggressive biological behavior of HER2+ and TN DCIS. We demonstrated that younger patients with p53 positive HER2+ DCIS had significantly adverse clinical outcome.

A probable shark dorsal fin spine fragment from the Early Triassic of the Arrow Rocks sequence, Whangaroa, northern New Zealand

The ornament on a small external cast in pink chert shows considerable similarity with that of various Middle Palaeozoic and Triassic fish genera. It comes from the Permian–Triassic Oruatemanu Formation of Arrow Rocks, Whangaroa area, eastern Northland. Conodont faunas from a few metres above and below the sample allow correlation with the Neospathodus pakistanensis zone of the Early Triassic, which is assigned to the late Dienerian (late Induan), with adjacent conodont zone faunas in their correct stratigraphic association. The cast is assumed to be that of a small fragment of fin spine, most likely from the junction area of the crown and root on the right-hand side of a dorsal fin spine, possibly anterior, of a marine ctenacanthoid shark, a basal shark order not previously recorded from New Zealand.