Spatial Overlap of Claudin- and Phosphatidylinositol Phosphate-Binding Sites on the First PDZ Domain of Zonula Occludens 1 Studied by NMR.

: The tight junction is an intercellular adhesion complex composed of claudins (CLDs), occludin, and the scaffolding proteins zonula occludens 1 (ZO-1) and its two paralogs ZO-2 and ZO-3. ZO-1 is a multifunctional protein that contains three PSD95/Discs large/ZO-1(PDZ) domains. A key functional domain of ZO-1 is the first PDZ domain (ZO-1(PDZ1)) that recognizes the conserved C-termini of CLDs.

Pharmacological induction of heat shock proteins ameliorates toxicity of mutant PKCγ in spinocerebellar ataxia type 14.

Amyloid and amyloid-like protein aggregations are hallmarks of multiple, varied neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. We previously reported that spinocerebellar ataxia type 14 (SCA14), a dominant-inherited neurodegenerative disease that affects cerebellar Purkinje cells, is characterized by the intracellular formation of neurotoxic amyloid-like aggregates of genetic variants of protein kinase Cγ (PKCγ). A number of protein chaperones, including heat shock protein 70 (Hsp70), promote the degradation and/or refolding of misfolded proteins and thereby prevent their aggregation.

Heat-induced native dimerization prevents amyloid formation by variable domain from immunoglobulin light-chain REI.

Unlabelled: Amyloid light-chain (AL) amyloidosis is a protein-misfolding disease characterized by accumulation of immunoglobulin light chains (LCs) into amyloid fibrils. Dimerization of a full length or variable domain (V ) of LC serves to stabilize the native state and prevent the formation of amyloid fibrils. We here analyzed the thermodynamic properties of dimerization and unfolding reactions by nonamyloidogenic V from REI LC or its monomeric Y96K mutant using sedimentation velocity and circular dichroism.

Cutting Edge: Class II-like Structural Features and Strong Receptor Binding of the Nonclassical HLA-G2 Isoform Homodimer.

HLA-G is a natural tolerogenic molecule and has the following unique features: seven isoforms (HLA-G1 to HLA-G7), formation of disulfide-linked homodimers, and β2-microglobulin (β2m)-free forms. Interestingly, individuals null for the major isoform, HLA-G1, are healthy and expressed the α2 domain-deleted isoform, HLA-G2, which presumably compensates for HLA-G1 function. However, the molecular characteristics of HLA-G2 are largely unknown.

Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques.

Protein folding is a thermodynamic process driven by energy gaps between the native and unfolded states. Although a wealth of information is available on the structure of folded species, there is a paucity of data on unfolded species. Here, we analyzed the structural properties of the unfolded state of the starch-binding domain of glucoamylase from Aspergillus niger (SBD) formed in the presence of guanidinium hydrochloride (GuHCl).

Crystal Structure of Human Herpesvirus 6B Tegument Protein U14.

The tegument protein U14 of human herpesvirus 6B (HHV-6B) constitutes the viral virion structure and is essential for viral growth. To define the characteristics and functions of U14, we determined the crystal structure of the N-terminal domain of HHV-6B U14 (U14-NTD) at 1.85 Å resolution.

Identification and characterization of PKCγ, a kinase associated with SCA14, as an amyloidogenic protein.

Amyloid assemblies are associated with a wide range of human disorders, including Alzheimer's and Parkinson's diseases. Here, we identify protein kinase C (PKC) γ, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKCγ in cultured cells, as well as in vitro incubation of PKCγ without heat or chemical denaturants, causes amyloid-like fibril formation of this protein.

Decreased amyloidogenicity caused by mutational modulation of surface properties of the immunoglobulin light chain BRE variable domain.

Amyloid formation by immunoglobulin light chain (LC) proteins is associated with amyloid light chain (AL) amyloidosis. Destabilization of the native state of the variable domain of the LC (VL) is known to be one of the critical factors in promoting the formation of amyloid fibrils. However, determining the key residues involved in this destabilization remains challenging, because of the existence of a number of intrinsic sequence variations within VL.

Structural basis of α-catenin recognition by EspB from enterohaemorrhagic E. coli based on hybrid strategy using low-resolution structural and protein dissection.

Enterohaemorrhagic E. coli (EHEC) induces actin reorganization of host cells by injecting various effectors into host cytosol through type III secretion systems. EspB is the natively partially folded EHEC effector which binds to host α-catenin to promote the actin bundling.

Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion.

Although the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion.

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60.

Binding of islet amyloid polypeptide to supported lipid bilayers and amyloid aggregation at the membranes.

Amyloid deposition of human islet amyloid polypeptide (hIAPP) in the islets of Langerhans is closely associated with the pathogenesis of type II diabetes mellitus. Despite substantial evidence linking amyloidogenic hIAPP to loss of β-cell mass and decreased pancreatic function, the molecular mechanism of hIAPP cytotoxicity is poorly understood. We here investigated the binding of hIAPP and nonamyloidogenic rat IAPP to substrate-supported planar bilayers and examined the membrane-mediated amyloid aggregation.

Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism.

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin.

N-terminal phosphorylation of HP1{alpha} promotes its chromatin binding.

The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli.

Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: a case for EspB as an intrinsically less-ordered effector.

Enterohemorrhagic and enteropathogenic Escherichia coli produce various effector proteins that are directly injected into the host-cell cytosol through the type III secretion system. E. coli secreted protein (Esp)B is one such effector protein, and affects host-cell morphology by reorganizing actin networks.

Structure and functional characterization of Vibrio parahaemolyticus thermostable direct hemolysin.

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.

Effect of lipid type on the binding of lipid vesicles to islet amyloid polypeptide amyloid fibrils.

Amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are observed near pancreatic beta-cells of type II diabetics, with their presence strongly correlating with a loss of beta-cell mass and decreased pancreatic function. Although beta-cell membranes have been implicated as the likely target of amyloidogenic IAPP toxicity, interactions between membranes and IAPP in the fibrillar state have not been well characterized. In this study, turbidity measurements were conducted to provide a detailed description of the binding reaction between IAPP fibrils and lipid vesicles made from phosphatidylcholine.

Competition between folding, native-state dimerisation and amyloid aggregation in beta-lactoglobulin.

We show that a series of peptides corresponding to individual beta-strands in native beta-lactoglobulin readily form amyloid aggregates and that such aggregates are capable of seeding fibril formation by a full-length form of beta-lactoglobulin in which the disulfide bonds are reduced. By contrast, preformed fibrils corresponding to only one of the beta-strands that we considered, betaA, were found to promote fibril formation by a full-length form of beta-lactoglobulin in which the disulfide bonds are intact. These results indicate that regions of high intrinsic aggregation propensity do not give rise to aggregation unless at least partial unfolding takes place.

Molecular basis of actin reorganization promoted by binding of enterohaemorrhagic Escherichia coli EspB to alpha-catenin.

EspB is a multifunctional protein associated with the type III secretion system of enterohaemorrhagic Escherichia coli, and interacts with various biomolecules including alpha-catenin in the host cell. The binding of EspB to alpha-catenin is thought be involved in actin reorganization during bacterial infection, although the precise mechanism of this phenomenon is still unclear. Recent research shows that dimerization of alpha-catenin dissociates it from E-cadherin/beta-catenin/alpha-catenin complexes, and that the dimer suppresses Arp2/3-mediated actin branching or polymerization.

Effect of an amyloidogenic sequence attached to yellow fluorescent protein.

Green fluorescent protein (GFP) is often misfolded into nonfluorescent states when an aggregatable sequence is attached to its N-terminus. However, GFP fusions with highly aggregatable, prion-determining, and highly charged sequences from yeast prions, such as Sup35 and Ure2p, form green fibrils with properly folded GFP. To gain further insight into the general effect of an aggregatable sequence attached to fluorescent protein, we designed eight fusion proteins of a yellow variant of GFP (YFP) containing an aggregation-prone amyloidogenic sequence derived from human medin, attached via different lengths of linker sequence.

Amyloid fibril formation and chaperone-like activity of peptides from alphaA-crystallin.

AlphaA-crystallin (alphaAC), a major component of eye lens, exhibits chaperone-like activity and is responsible for maintaining eye lens transparency. Synthetic peptides which corresponded to the putative substrate-binding site of alphaAC have been reported to prevent aggregation of proteins [Sharma, K. K.

Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy.

The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents.

Expression and purification of recombinant human annexin A2 in Pichia pastoris and utility of expression product for detecting annexin A2 antibody.

Annexin A2, a Ca2+-dependent phospholipid binding protein, is abundantly expressed in various human organs, which exists as either a membrane-associated, cytosolic or soluble form in serum. We constructed expression systems for recombinant human annexin A2 (rhA2) using Pichia pastoris. The systems are designed to secrete rhA2 as either the N- or C-terminally His6-tagged form to facilitate purification.

Thermostable direct hemolysin of Vibrio parahaemolyticus is a bacterial reversible amyloid toxin.

Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, is detoxified by heating at approximately 60-70 degrees C but is reactivated by additional heating above 80 degrees C. This paradoxical phenomenon, known as the Arrhenius effect, has remained unexplained for approximately 100 years. We now demonstrate that the effect is related to structural changes in the protein that produce fibrils.