Metal Ion-dependent Effects of Clioquinol on the Fibril Growth of an Amyloid β Peptide

This research was originally published in the Journal of Biological Chemistry. Bakthisaran Raman, Tadato Ban, Kei-ichi Yamaguchi, Miyo Sakai, Tomoji Kawai, Hironobu Naiki and Yuji Goto. Metal Ion-dependent Effects of Clioquinol on the Fibril Growth of an Amyloid β Peptide. J. Biol. Chem. 2005; 280, 16157-16162. © the American Society for Biochemistry and Molecular Biolog

Real-time and Single Fibril Observation of the Formation of Amyloid β Spherulitic Structures

This research was originally published in the Journal of Biological Chemistry. Tadato Ban, Kenichi Morigaki, Hisashi Yagi, Takashi Kawasaki, Atsuko Kobayashi, Shunsuke Yuba, Hironobu Naiki and Yuji Goto. Real-time and Single Fibril Observation of the Formation of Amyloid β Spherulitic Structures. J. Biol. Chem. 2006; 281, 33677–33683. © the American Society for Biochemistry and Molecular Biolog

OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation

The dynamin-related GTPase OPA1 is mutated in autosomal dominant optic atrophy (DOA) (Kjer type), an inherited neuropathy of the retinal ganglion cells. OPA1 is essential for the fusion of the inner mitochondrial membranes, but its mechanism of action remains poorly understood. Here we show that OPA1 has a low basal rate of GTP hydrolysis that is dramatically enhanced by association with liposomes containing negative phospholipids such as cardiolipin. Lipid association triggers assembly of OPA1 into higher order oligomers. In addition, we find that OPA1 can promote the protrusion of lipid tubules from the surface of cardiolipin-containing liposomes. In such lipid protrusions, OPA1 assemblies are observed on the outside of the lipid tubule surface, a protein-membrane topology similar to that of classical dynamins. The membrane tubulation activity of OPA1 is suppressed by GTPγS. OPA1 disease alleles associated with DOA display selective defects in several activities, including cardiolipin association, GTP hydrolysis and membrane tubulation. These findings indicate that interaction of OPA1 with membranes can stimulate higher order assembly, enhance GTP hydrolysis and lead to membrane deformation into tubules

Direct Observation of Amyloid Fibril Growth Monitored by Thioflavin T Fluorescence

This research was originally published in the Journal of Biological Chemistry. Tadato Ban, Daizo Hamada, Kazuhiro Hasegawa, Hironobu Naiki and Yuji Goto. Direct Observation of Amyloid Fibril Growth Monitored by Thioflavin T Fluorescence. J. Biol. Chem. 2003; 278, 16462-16465. © the American Society for Biochemistry and Molecular Biolog

Destruction of Amyloid Fibrils of a β2-Microglobulin Fragment by Laser Beam Irradiation

This research was originally published in the Journal of Biological Chemistry. Daisaku Ozawa, Hisashi Yagi, Tadato Ban, Atsushi Kameda, Toru Kawakami, Hironobu Naiki and Yuji Goto. Destruction of Amyloid Fibrils of a β2-Microglobulin Fragment by Laser Beam Irradiation. J. Biol. Chem. 2009; 284, 1009-1017. © the American Society for Biochemistry and Molecular Biolog

Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology(1,2). Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy(2-4). However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans-OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control

Branching in amyloid fibril growth

AbstractUsing the peptide hormone glucagon and Aβ(1–40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35–40°. Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Aβ(1–40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Aβ(1–40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growt

Critical balance of electrostatic and hydrophobic interactions is required for β<SUB>2</SUB>-Microglobulin amyloid fibril growth and stability

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO<SUB>4</SUB>, and Na<SUB>2</SUB>SO<SUB>4</SUB> on the amyloid fibril growth of &#946;<SUB>2</SUB>-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO<SUB>4</SUB><SUP>2-</SUP> > ClO<SUB>4</SUB><SUP>-</SUP> > I<SUP>-</SUP> > Cl<SUP>-</SUP>, consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of &#946;<SUB>2</SUB>-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance