Effects of bound nucleotides on the secondary structure, thermal stability, and phosphorylation of Rab3A.

Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2.

LRRK2-mediated phosphorylation and thermal stability of Rab12 are regulated by bound nucleotides.

An abnormal increase in the phosphorylation of Rab12 by leucine-rich repeat kinase 2 (LRRK2), a serine/threonine kinase genetically linked to Parkinson's disease (PD), has been implicated in the pathogenesis of PD, although the underlying mechanism remains unclear. In this report, we show that LRRK2 phosphorylates Rab12 more efficiently in its GDP-bound form than in its GTP-bound form using an in vitro phosphorylation assay. This observation suggests that LRRK2 recognizes the structural difference of Rab12 caused by the bound nucleotide and that Rab12 phosphorylation inhibits its activation. Circular dichroism data revealed that Rab12, in its GDP-bound form, is more susceptible to heat-induced denaturation than its GTP-bound form, which was exacerbated at basic pH. Differential scanning fluorimetry showed that heat-induced denaturation of Rab12 in its GDP-bound form occurs at a lower temperature than in its GTP-bound form. These results suggest that the type of nucleotide bound to Rab12 determines the efficiency of LRRK2-mediated phosphorylation and the thermal stability of Rab12, and provide insights into elucidating the mechanism of the abnormal increase in Rab12 phosphorylation.

Overview of the Impact of Pathogenic LRRK2 Mutations in Parkinson's Disease.

Leucine-rich repeat kinase 2 (LRRK2) is a large protein kinase that physiologically phosphorylates and regulates the function of several Rab proteins. LRRK2 is genetically implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD), although the underlying mechanism is not well understood. Several pathogenic mutations in the LRRK2 gene have been identified, and in most cases the clinical symptoms that PD patients with LRRK2 mutations develop are indistinguishable from those of typical PD. However, it has been shown that the pathological manifestations in the brains of PD patients with LRRK2 mutations are remarkably variable when compared to sporadic PD, ranging from typical PD pathology with Lewy bodies to nigral degeneration with deposition of other amyloidogenic proteins. The pathogenic mutations in LRRK2 are also known to affect the functions and structure of LRRK2, the differences in which may be partly attributable to the variations observed in patient pathology. In this review, in order to help researchers unfamiliar with the field to understand the mechanism of pathogenesis of LRRK2-associated PD, we summarize the clinical and pathological manifestations caused by pathogenic mutations in LRRK2, their impact on the molecular function and structure of LRRK2, and their historical background.

A tailored tetravalent peptide displays dual functions to inhibit amyloid ß production and aggregation.

Inhibition of amyloid-ß peptide (Aß) accumulation in the brain is a promising approach for treatment of Alzheimer's disease (AD). Aß is produced by ß-secretase and γ-secretase in endosomes via sequential proteolysis of amyloid precursor protein (APP). Aß and APP have a common feature to readily cluster to form multimers. Here, using multivalent peptide library screens, we identified a tetravalent peptide, LME-tet, which binds APP and Aß via multivalent interactions. In cells, LME-tet-bound APP in the plasma membrane is transported to endosomes, blocking Aß production through specific inhibition of ß-cleavage, but not γ-cleavage. LME-tet further suppresses Aß aggregation by blocking formation of the ß-sheet conformation. Inhibitory effects are not observed with a monomeric peptide, emphasizing the significance of multivalent interactions for mediating these activities. Critically, LME-tet efficiently reduces Aß levels in the brain of AD model mice, suggesting it may hold promise for treatment of AD.

Site-specific amino acid D-isomerization of Tau R2 and R3 peptides changes the fibril morphology, resulting in attenuation of Tau aggregation inhibitor potency.

Tau, a microtubule-binding protein, is a major component of neurofibrillary tangles in the brains of Alzheimer's disease patients. Tau aggregation following fibril formation induces Alzheimer's disease pathogenesis. The accumulation of D-isomerized amino acids in proteins that occurs in several tissues with aging is thought to be implicated in age-related diseases. D-isomerized Asp accumulation has also been found in Tau in neurofibrillary tangles. We previously demonstrated the effects of D-isomerization of Asp within microtubule-binding repeat peptides of Tau, Tau R2, and R3 on the rates of structural transition and fibril formation. Here, we investigated the potency of Tau aggregation inhibitors on fibril formation of wild-type Tau R2 and R3 peptides and D-isomerized Asp-containing Tau R2 and R3 peptides. D-isomerization of Asp within Tau R2 and R3 peptides attenuated the potency of inhibitors. We next investigated the fibril morphology of D-isomerized Asp-containing Tau R2 and R3 peptides by electron microscopy. D-isomerized Asp-containing Tau R2 and R3 fibrils showed significantly different fibril morphology from that of wild-type peptides. Our results indicate that D-isomerization of Asp within Tau R2 and R3 peptides affects fibril morphology, resulting in attenuation of the potency of Tau aggregation inhibitors.

Physicochemical characterization of the G51D mutation of α-synuclein that is responsible for its severe cytotoxicity.

Fibril formation and aggregation of α-synuclein are important for the pathogenesis of neurodegenerative disorders including Parkinson's disease. In familial Parkinson's disease, the G51D mutation of α-synuclein causes severe symptoms and rapid progression. α-Synuclein, an intrinsically disordered protein, was shown to adopt an α-helical tetrameric state that resists fibrillation and aggregation. Here, we isolated the stable dimeric state of recombinant wild-type (WT) α-synuclein and G51D α-synuclein protein. Using circular dichroism spectroscopy, we determined that the α-synuclein dimer and monomer structures were unfolded. The WT α-synuclein dimer was more resistant to fibril formation than the monomer. However, the fibril formation rate of the G51D α-synuclein dimer was similar to that of the G51D α-synuclein monomer. The fibril morphology and properties of the G51D α-synuclein monomer were different from those of the WT α-synuclein monomer and dimer and G51D α-synuclein dimer. Additionally, G51D α-synuclein monomer fibrils were more cytotoxic than other fibrils. Our findings indicate that the structural differences between G51D α-synuclein monomer fibrils and other fibrils are critically responsible for its severe neurotoxicity in familial Parkinson's disease.

The dynamic structure of Rab35 is stabilized in the presence of GTP under physiological conditions.

Rab proteins, a family of small guanosine triphosphatases, play key roles in intracellular membrane trafficking and the regulation of various cellular processes. As a Rab isoform, Rab35 is crucial for recycling endosome trafficking, cytokinesis and neurite outgrowth. In this report, we analyzed dynamic structural changes and physicochemical features of Rab35 in response to different external conditions, including temperature, pH, salt concentration and guanosine triphosphate (GTP), by circular dichroism (CD) spectroscopy. CD spectra revealed that the α-helix content of Rab35 varies under different conditions considerably. The addition of GTP increases the α-helix content of Rab35 when the temperature, pH and salt concentration match physiological conditions. The results suggest that the external environment affects the secondary structure of Rab35. In particular, the presence of GTP stabilized the α-helices of Rab35 under physiological conditions. These structural changes may translate to changes in Rab35 function and relate to its role in membrane trafficking.

Effect of site-specific amino acid D-isomerization on ß-sheet transition and fibril formation profiles of Tau microtubule-binding repeat peptides.

d-amino acid-containing proteins have been found in several human tissues, and the spontaneous accumulation of d-amino acids in proteins is thought to be involved in age-dependent diseases including dementia. Tau, a microtubule-associated protein, is a major component of neurofibrillary tangles in Alzheimer's disease. Site-specific amino acid D-isomerization in Tau has been observed in the brains of patients with Alzheimer's disease. Here, we conducted amino acid D-isomerization at specific sites in microtubule-binding repeat peptides of Tau (Tau R2 and R3) and examined the effects on Tau structure and fibril formation. Our results demonstrate that amino acid D-isomerization in Tau R2 peptides decreased the rates of ß-sheet transition and fibril formation compared with those of the wild-type peptide composed of all l-amino acids. In contrast, Tau R3 peptides that had undergone amino acid D-isomerization at either Asp314, Ser316, or Ser324 showed increased rates of ß-sheet transition and fibril formation compared with those of the wild-type Tau R3 peptide.

The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation.

In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.

A docking model of dapsone bound to HLA-B*13:01 explains the risk of dapsone hypersensitivity syndrome.

BACKGROUND: Dapsone (4,4'-diaminodiphenylsulfone) has been widely used for the treatment of infections such as leprosy. Dapsone hypersensitivity syndrome (DHS) is a major side effect, developing in 0.5-3.6% of patients treated with dapsone, and its mortality rate is ∼10%. Recently, human leukocyte antigen (HLA)-B*13:01 was identified as a marker of susceptibility to DHS. OBJECTIVES: To investigate why HLA-B*13:01 is responsible for DHS from a structural point of view. METHODS: First, we used homology modeling to derive the three-dimensional structures of HLA-B*13:01 (associated with DHS) and HLA-B*13:02 (not so associated despite strong sequence identity [99%] with HLA-B*13:01). Next, we used molecular docking, molecular dynamic simulations, and the molecular mechanics Poisson-Boltzman surface area method, to investigate the interactions of dapsone with HLA-B*13:01 and 13:02. RESULTS: We found a crucial structural difference between HLA-B*13:01 and 13:02 in the F-pocket of the antigen-binding site. As Trp95 in the α-domain of HLA-B*13:02 is replaced with the less bulky Ile95 in HLA-B*13:01, we found an additional well-defined sub-pocket within the antigen-binding site of HLA-B*13:01. All three representative docking poses of dapsone against the antigen-binding site of HLA-B*13:01 used this unique sub-pocket, indicating its suitability for binding dapsone. However, HLA-B*13:02 does not seem to possess a binding pocket suitable for binding dapsone. Finally, a binding free energy calculation combined with a molecular dynamics simulation and the molecular mechanics Poisson-Boltzman surface area method indicated that the binding affinity of dapsone for HLA-B*13:01 would be much greater than that for HLA-B*13:02. CONCLUSIONS: Our computational results suggest that dapsone would fit within the structure of the antigen-recognition site of HLA-B*13:01. This may change the self-peptides that bind to HLA-B*13:01, explaining why HLA-B*13:01 is a marker of DHS susceptibility.

Phosphorylation of syntaxin-3 at Thr 14 negatively regulates exocytosis in RBL-2H3 mast cells.

Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2.

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins.

Mast cells are involved in allergic responses and undergo exocytotic release of inflammatory mediators in response to antigen stimulation. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this membrane fusion process; some SNARE-binding proteins regulate SNARE-dependent liposome membrane fusion. SNARE-binding protein complexin II is expressed in mast cells, where it positively regulates exocytotic release after antigen stimulation. We found that complexin II suppressed SNARE-dependent membrane fusion between mast cell SNARE-containing liposomes. This inhibitory effect of complexin II was abolished when we used a structurally divergent mutant (R59H) complexin II, where Arg59 is substituted with histidine. These results suggest that complexin II negatively regulates SNARE-dependent exocytotic membrane fusion in mast cells, and this inhibitory effect is dependent upon Arg59.

Allosteric Breakage of the Hydrogen Bond within the Dual-Histidine Motif in the Active Site of Human Pin1 PPIase.

Intimate cooperativity among active site residues in enzymes is a key factor for regulating elaborate reactions that would otherwise not occur readily. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is the phosphorylation-dependent cis-trans peptidyl-prolyl isomerase (PPIase) that specifically targets phosphorylated Ser/Thr-Pro motifs. Residues C113, H59, H157, and T152 form a hydrogen bond network in the active site, as in the noted connection. Theoretical studies have shown that protonation to thiolate C113 leads to rearrangement of this hydrogen bond network, with switching of the tautomeric states of adjacent histidines (H59 and H157) [Barman, A., and Hamelberg, D. (2014) Biochemistry 53, 3839-3850]. This is called the "dual-histidine motif". Here, C113A and C113S Pin1 mutants were found to alter the protonation states of H59 according to the respective residue type replaced at C113, and the mutations resulted in disruption of the hydrogen bond within the dual-histidine motif. In the C113A mutant, H59 was observed to be in exchange between ε- and δ-tautomers, which widened the entrance of the active site cavity, as seen by an increase in the distance between residues A113 and S154. The C113S mutant caused H59 to exchange between the ε-tautomer and imidazolium while not changing the active site structure. Moreover, the imidazole ring orientations of H59 and H157 were changed in the C113S mutant. These results demonstrated that a mutation at C113 modulates the hydrogen bond network dynamics. Thus, C113 acts as a pivot to drive the concerted function among the residues in the hydrogen bond network, as theoretically predicted.

The C113D mutation in human Pin1 causes allosteric structural changes in the phosphate binding pocket of the PPIase domain through the tug of war in the dual-histidine motif.

Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.

Site-specific aspartic acid isomerization regulates self-assembly and neurotoxicity of amyloid-ß.

Amyloid-ß (Aß) proteins, which consist of 42 amino acids (Aß1­42), are the major constituent of neuritic plaques that form in the brains of senile patients with Alzheimer's disease (AD). Several reports state that three aspartic acid (Asp) residues at positions 1, 7, and 23 in Aß1­42 in the plaques of patients with AD are highly isomerized from the L- to D-form. Using biophysical experiments, the present study shows that simultaneous D-isomerization of Asp residues at positions 7 and 23 (D-Asp(7,23)) enhances oligomerization, fibril formation, and neurotoxic effect of Aß1­42. In addition, D-isomerization of Asp at position 1 (D-Asp(1)) suppresses malignant effects induced by D-Asp(7,23) of Aß1­42. These results provide fundamental information to elucidate molecular mechanisms of AD pathogenesis and to develop potent inhibitors of amyloid aggregates and Aß neurotoxicity.

Tetrameric interaction of the ectoenzyme CD38 on the cell surface enables its catalytic and raft-association activities.

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.

D-Ser-containing humanin shows promotion of fibril formation.

Humanin (HN), a peptide of 24 amino acid residues, suppresses the neuronal cell death that is induced by the gene products of Alzheimer's disease. HN contains two Ser residues at positions 7 and 14. Because the proportion of D-Ser isomerized from L-Ser in proteins appears to increase as cellular organs age, we explored the structural effects of the isomerization of each Ser residue in HN. By using a thioflavin-T assay to detect fibril formation, we found that an HN derivative that contained two isomerized D-Ser residues had a greater tendency to form fibrils than did wild-type HN or HNs containing single D-Ser residues. A previous report showed that HN containing two D-Ser residues exerts neuroprotective activity. Our data, therefore, suggest that the fibril formation by HN that contains two D-Ser residues may promote HN neuroprotective activity.

Controlled structure and properties of silicate nanoparticle networks for incorporation of biosystem components.

Inorganic nanoparticles are of technological interest in many fields. We created silicate nanoparticle hydrogels that effectively incorporated biomolecules that are unstable and involved in complicated reactions. The size of the silicate nanoparticles strongly affected both the physical characteristics of the resulting hydrogel and the activity of biomolecules incorporated within the hydrogel. We used high-resolution transmission electron microscopy (TEM) to analyze in detail the hydrogel network patterns formed by the silicate nanoparticles. We obtained clear nanostructured images of biomolecule-nanoparticle composite hydrogels. The TEM images also showed that larger silicate nanoparticles (22 nm) formed more loosely associated silicate networks than did smaller silicate nanoparticles (7 nm). The loosely associated networks formed from larger silicate nanoparticles might facilitate substrate diffusion through the network, thus promoting the observed increased activity of the entrapped biomolecules. This doubled the activity of the incorporated biosystems compared with that of biosystems prepared by our own previously reported method. We propose a reaction scheme to explain the formation of the silicate nanoparticle networks. The successful incorporation of biomolecules into the nanoparticle hydrogels, along with the high level of activity exhibited by the biomolecules required for complicated reaction within the gels, demonstrates the nanocomposites' potential for use in medical applications.

Stability of folding structure of Zic zinc finger proteins.

Zic family proteins have five C(2)H(2)-type zinc finger (ZF) motifs. We physicochemically characterized the folding properties of Zic ZFs. Alteration of chelation with zinc ions and of hydrophobic interactions changed circular dichroism spectra, suggesting that they caused structural changes. The motifs were heat stable, but electrostatic interactions had little effect on structural stability. These results highlight the importance of chelating interactions and hydrophobic interactions for the stability of the folding structure of Zic ZF proteins.

Screening system for D-Asp-containing proteins using D-aspartyl endopeptidase and two-dimensional gel electrophoresis.

D-Asp-containing proteins have been implicated in many aging-related diseases. To clarify the role of D-Asp-containing proteins in such diseases, we developed a screening system for these proteins using a D-aspartyl endopeptidase that specifically cleaves the proteins at the C-terminus. The digested proteins were detected by means of two-dimensional gel electrophoresis and identified using nano-liquid chromatography/tandem mass spectrometry. We were able to detect myelin basic protein, a known D-Asp-containing protein, in the brain tissues of mice; this indicates that our system is effective for screening D-Asp-containing proteins.