Nitrosative stress has recently been demonstrated as a causal in a select sporadic variant of Parkinson's (PD) and Alzheimer's (AD) diseases. Specifically, elevated levels of NO disrupt the redox activity of protein-disulfide isomerase, a key endoplasmic reticulum-resident chaperone by S-nitroso modification of its redox-active cysteines. This leads to accumulation of misfolded AD- and PD-specific protein debris. We have recently demonstrated in vitro that polyphenolic phytochemicals, curcumin and masoprocol, can rescue S-nitroso-PDI formation by scavenging NOx. In this study, using dopaminergic SHSY-5Y cells, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1 (a known constituent of PD Lewy neurites) as a function of rotenone-induced nitrosative stress. Importantly, we demonstrate a marked decrease in synphilin-1 aggregation when the cell line is previously incubated with 3,5-bis(2-flurobenzylidene) piperidin-4-one (EF-24), a curcumin analogue, prior to rotenone insult. Furthermore, our data also reveal that rotenone attenuates PDI expression in the same cell line, a phenomenon that can be mitigated through EF-24 intervention. Together, these results suggest that EF-24 can exert neuroprotective effects by ameliorating nitrosative stress-linked damage to PDI and the associated onset of PD and AD. Essentially, EF-24 can serve as a scaffold for the design and development of PD and AD specific prophylactics.

Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

A key feature of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders including Alzheimer disease (AD), Parkinson disease (PD) and Huntington's disease (HD) is abnormal aggregation and deposition of misfolded proteins. Previous studies have shown that autophagy plays an important role in the clearance of disease-linked protein aggregates. In the current study, we report that ibudilast, which is a non-selective inhibitor of phosphodiesterases (PDEs) and an anti-inflammation drug, can induce autophagy and lysosomal biogenesis through mammalian target of rapamycin complex 1 - transcription factor EB (mTORC1-TFEB) signaling. We have found that ibudilast significantly enhances the clearance of disease-linked TAR DNA binding protein (TDP-43) and superoxide dismutase 1 (SOD1) protein aggregates in transfected cellular models carrying corresponding gene mutations. The mechanistic study revealed that ibudilast could markedly enhance TFEB nuclear translocation and increase the autolysosomes by inhibiting mTORC1 activity. We have also demonstrated that ibudilast could protect TDP-43-induced cytotoxicity in motor neuron-like NSC-34 cells. Collectively, our study identifies ibudilast as an autophagy enhancer and provides insights into the molecular basis of ibudilast for the potential treatment of several neurodegenerative disorders.

Introduction of carbonyl groups into amino acid residues is a hallmark for oxidative damage to proteins by reactive oxygen species (ROS). Protein carbonylation can have deleterious effects on cell function and viability, since it is generally unrepairable by cells and can lead to protein dysfunction and to the production of potentially harmful protein aggregates. Meanwhile, pyridoxamine (PM) is known to scavenge various toxic carbonyl species derived from either glucose or lipid degradation through nucleophilic addition. PM is also demonstrated to catalyze non-enzymatic transamination reactions between amino and α-keto acids. Here, we found that PM scavenges protein carbonyls in oxidized BSA with concomitant generation of pyridoxal and recovers oxidized lysozyme activity. Moreover, we demonstrated that the treatment of H2O2-exposed HepG2 hepatocytes with PM significantly reduced levels of cellular carbonylated proteins and aggregated proteins, and also improved cell survival rate. Our results suggest that PM may have potential efficacy in ameliorating ROS-mediated cellular dysfunction.

Using solvent-exposed intramolecular backbone hydrogen bonds as physico-chemical descriptors for protein packing, a role for transient, non-obligate oligomers in the formation of aberrant protein aggregates is presented. Oligomeric models of the both wild type (wt) and select mutant variants of superoxide dismutase (SOD1) are proposed to provide a structural basis for investigating the etiology of Amyotrophic Lateral Sclerosis (ALS).

Historically, the two most prominent proteins in Alzheimer's disease (AD) research have been the amyloid precursor protein (APP) and the microtubule assembly protein tau. In the classical model for the etiology of AD, amyloid-β (Aβ)-an APP derivative and hyperphosphorylated tau form aggregates in the brain that underlie the pathogenesis of the disease. However, the connection between Aβ and tau pathologies remains unclear. Several studies have provided evidence that the presence of Aβ can induce or enhance neurofibrillary tangle formation by tau. Others have reported a direct interaction between tau and short fragments of the APP transmembrane domain, C99. Structural studies of C99 show that these in vitro tau-binding fragments of C99 are buried in the lipid bilayer and are likely unavailable to bind tau in vivo. Given the importance of APP and tau in AD, we sought to characterize the potential interaction of the Aβ precursor, full length C99, and tau in vitro using NMR spectroscopy. We found that C99 and soluble tau interact only weakly and, most likely, non-specifically.

Junctophilin-2 (JPH2) was conventionally considered as a structural membrane binding protein. Recently, it was shown that proteolytically truncated mouse JPH2 variants are imported into nucleus to exert alternative functions. However, the intranuclear behaviors of human JPH2 (hJPH2) and underlying molecular determinants have not been explored. Here, we demonstrate that full-length hJPH2 is imported into nucleus in human cells by two nuclear localization signals (NLSs), including a newly discovered one at the C-terminus. Importantly, unlike the JPH2 N-terminal truncation which diffuses throughout the nucleus, full-length hJPH2 forms nuclear bodies behaving like liquid-liquid phase separated droplets that are separated from chromatin. The C-terminal transmembrane domain is required for the formation of hJPH2 droplets. Oxidation mimicking substitution of residues C678 and M679 augments the formation of hJPH2 nuclear droplets, suggesting nuclear hJPH2 liquid-liquid phase separation could be modulated by oxidative stress. Mutation A405D, which introduces a negatively charged residue into an intrinsic disordered region (IDR) of hJPH2, turns liquid-like droplets into amyloid-like aggregates. Depletion of an Alanine Rich Region in the IDR recapitulates the liquid-amyloid phase transition. The MORN repeat regions of hJPH2 encodes intrinsic tendency to form amyloid-like structure. Together, these data revealed the novel intrinsic properties of hJPH2 to form nuclear liquid droplets, and identified critical functional domains encoding these properties. We propose that hJPH2 droplets could function as membrane-less organelles participating in nuclear regulatory processes.

Mutations in protein kinase Cgamma (PKCgamma) cause the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14). In this study, expression of an extensive panel of known SCA14-associated PKCgamma mutations as fusion proteins in cell culture led to the consistent formation of cytoplasmic aggregates in response to purinoceptor stimulation. Aggregates co-stained with antibodies to phosphorylated PKCgamma and the early endosome marker EEA1 but failed to redistribute to the cell membrane under conditions of oxidative stress. These studies suggest that Purkinje cell damage in SCA14 may result from a reduction of PKCgamma activity due its aberrant sequestration in the early endosome compartment.

Paget's disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone resorption, with osteoclasts that are increased in size, multinuclearity, number and activity. PDB-causing missense and nonsense variants in the gene encoding Sequestosome-1/p62 (SQSTM1) have been identified, all of which cluster in and around the ubiquitin-associated (UBA) domain of the protein. SQSTM1 is ubiquitously expressed and there is, as yet, no clear reason why these mutations only appear to cause an osteoclast-related phenotype. Using co-immunoprecipitation and tandem mass spectrometry, we identified a novel interaction in human osteoclast-like cells between SQSTM1 and Autophagy-Linked FYVE domain-containing protein (ALFY/WDFY3). Endogenous ALFY and SQSTM1 both localised within the nuclei of osteoclasts and their mononuclear precursors. When osteoclasts were starved to induce autophagy, SQSTM1 and ALFY relocated to the cytoplasm where they formed large aggregates, with cytoplasmic relocalisation appearing more rapid in mature osteoclasts than in precursors in the same culture. Overexpression of wild-type SQSTM1 in HEK293 cells also resulted in the formation of cytoplasmic aggregates containing SQSTM1 and endogenous ALFY, as did overexpression of a PDB-causing missense mutant form of SQSTM1, indicating that this mutation does not impair the formation of SQSTM1- and ALFY-containing aggregates. Expression of ALFY in bone cells has not previously been reported, and the process of autophagy has not been studied with respect to osteoclast activity. We have identified a functional interaction between SQSTM1 and ALFY in osteoclasts under conditions of cell stress. The difference in response to starvation between mature osteoclasts and their precursors may begin to explain the cell-specific functional effects of SQSTM1 mutations in PDB.

To investigate molecular mechanisms linking inflammation with neurodegeneration, we treated neuronal cultures with prostaglandins (PGs), which are mediators of inflammation. PGA1, D2, J2, and Delta12-PGJ2, but not PGE2, reduced the viability and raised the levels of ubiquitinated proteins in the neuronal cells. PGJ2 and its metabolite, Delta12-PGJ2, were the most potent of the four neurotoxic PGs tested in inducing both effects. To address the mechanism by which these agents lead to the accumulation of ubiquitinated proteins, we tested their effects on neuronal ubiquitin hydrolases UCH-L1 and UCH-L3 as well as on proteasome activity. Notably, Delta12-PGJ2 inhibited the activities of UCH-L1 (K(i) approximately 3.5 microM) and UCH-L3 (K(i) approximately 8.1 microM) without affecting proteasome activity. Intracellular aggregates containing ubiquitinated proteins were detected in Delta12-PGJ2-treated cells, indicating that these aggregates can form independently of proteasome inhibition. In conclusion, impairment of ubiquitin hydrolase activity, such as triggered by Delta12-PGJ2, may be an important contributor to neurodegeneration associated with accumulation of ubiquitinated proteins and inflammation.

Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC50 values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation.

Huntington's disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

In most cases high cytotoxicity is characteristic of aggregates formed during lag phase of amyloid formation, whereas mature fibrils represent the depot of protein molecules incapable of damaging cell membranes. However, new experimental data show that in cases of some proteins the fibrils are the most toxic type of aggregates. Meanwhile, structural characteristics of cytotoxic fibrils and mechanisms of their cell damaging action are insufficiently explored. This work is dedicated to studying amyloid aggregation of bovine carbonic anhydrase (BCA) and effect of aggregates formed at different stages of amyloid formation on viability of the cells. Here we demonstrate that oligomers formed during lag phase do not decrease cell viability, whereas protofibrils and amyloids of BCA are cytotoxic. Obtained results allow concluding that toxicity of BCA aggregates is associated with the presence of amyloid cross-β-structure, which signature is absorbance peak at low wavenumbers at FTIR spectra (1615-1630 cm-1). Our data suppose that cross-β-core of ВСА amyloid fibrils is responsible for their cytotoxicity.

Eukaryotic proteasome assembly is assisted by multiple dedicated chaperones. In yeast, formation of the heteroheptameric ring composed of α1-α7 subunits is promoted by the heterodimeric chaperone Pba3-Pba4. Here we reveal that in the absence of this dimeric chaperone, α2 replaces α4 during α-ring assembly, thereby giving rise to a non-productive complex that lacks α4, β1, β5, β6, and β7 subunits and aggregates of α4. Furthermore, our structure-guided mutational data demonstrate that the Pba3-Pba4 heterodimer acts as molecular matchmaker reinforcing the interaction between α4 and α5, which is the crucial step in the α-ring formation.

In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII.

Pathological aggregates of tau protein are found in several neurodegenerative diseases termed 'tauopathies'. Increasing evidence indicates that tau oligomer species rather than the large amyloid cytoplasmic inclusions relevant for histopathological diagnosis might be crucial for cellular damage and neurodegeneration. Trivalent metal ions and polyanionic structures like heparin or arachidonic acid have been shown to induce tau aggregation. However, little is known about early processes of tau aggregation. In this study, we applied fluorescence correlation spectroscopy (FCS) and scanning for intensely fluorescent targets (SIFT) to investigate oligomer formation of tau protein at nanomolar protein concentrations at the single-particle level. Our results indicate that the formation of distinct tau oligomers is induced by the trivalent metal ions Fe(3+) and Al(3+) and by organic solvents like DMSO, respectively. In contrast, bivalent metal ions (Cu(2+), Zn(2+), Mn(2+), Ca(2+), Mg(2+)) had no effect. While DMSO-induced small tau oligomers are relatively stable in solution, dynamic remodeling can be initiated by non-ionic detergents. Moreover Al(3+) induces rapid formation of a different oligomer species of larger size. Our results provide further insights into early tau oligomerization and aggregation dynamics.

Certain denatured proteins function as cofactors in the activation of plasminogen by tissue-type plasminogen activator. The present study approached the structural requirements for the cofactor activity of a model protein (human serum albumin). Heat denaturation of 100-230 microM albumin (80 degrees C and 60-90 min) reproducibly yielded aggregates with radius in the range of 10-150 nm. The major determinant of the cofactor potency was the size of the aggregates. The increase of particle size correlated with the cofactor activity, and there was a minimal requirement for the size of the cofactor (about 10 nm radius). Similar to other proteins, the molecular aggregates with cofactor function contained a significant amount of antiparallel intermolecular beta-sheets. Plasmin pre-digestion increased the cofactor efficiency (related to C-terminal lysine exposure) and did not affect profoundly the structure of the aggregates, suggesting a long-lasting and even a self-augmenting cofactor function of the denatured protein.

Transactive response DNA-binding protein of 43 kDa (TDP-43) abnormally forms aggregates in certain subtypes of frontotemporal lobar degeneration (FTLD) and in amyotrophic lateral sclerosis (ALS). The pathological forms of TDP-43 have reported to be associated with poly(ADP-ribose) (PAR), which regulates the properties of these aggregates. A recent study has indicated that tankyrase, a member of the PAR polymerase (PARP) family, regulates pathological TDP-43 formation under conditions of stress, and tankyrase inhibitors suppress TDP-43 aggregate formation and cytotoxicity. Since we reported the development of tankyrase inhibitors that are more specific than conventional inhibitors, in this study, we examined their effects on the formation of TDP-43 aggregates in cultured cells. Time-lapse imaging showed that TDP-43 aggregates appeared in the nucleus within 30 min of treatment with sodium arsenite. Several tankyrase inhibitors suppressed the formation of aggregates and decreased the levels of the tankyrase protein. Immunohistochemical studies demonstrated that tankyrase was localized to neuronal cytoplasmic inclusions in the spinal cords of patients with ALS. Moreover, the tankyrase protein levels were significantly higher in the brains of patients with FTLD than in the brains of control subjects. These findings suggest that the inhibition of tankyrase activity protects against TDP-43 toxicity. Tankyrase inhibitors may be a potential treatment to suppress the progression of TDP-43 proteinopathies.

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.