Transient sampling of aggregation‐prone conformations causes pathogenic instability of a parkinsonian mutant of DJ‐1 at physiological temperature

Various missense mutations in the cytoprotective protein DJ‐1 cause rare forms of inherited parkinsonism. One mutation, M26I, diminishes DJ‐1 protein levels in the cell but does not result in large changes in the three‐dimensional structure or thermal stability of the protein. Therefore, the molecular defect that results in loss of M26I DJ‐1 protective function is unclear. Using NMR spectroscopy near physiological temperature, we found that the picosecond–nanosecond dynamics of wild‐type and M26I DJ‐1 are similar. In contrast, elevated amide hydrogen/deuterium exchange rates indicate that M26I DJ‐1 is more flexible than the wild‐type protein on longer timescales and that hydrophobic regions of M26I DJ‐1 are transiently exposed to solvent. Tryptophan fluorescence spectroscopy and thiol crosslinking analyzed by mass spectrometry also demonstrate that M26I DJ‐1 samples conformations that differ from the wild‐type protein at 37°C. These transiently sampled conformations are unstable and cause M26I DJ‐1 to aggregate in vitro at physiological temperature but not at lower temperatures. M26I DJ‐1 aggregation is correlated with pathogenicity, as the structurally similar but non‐pathogenic M26L mutation does not aggregate at 37°C. The onset of dynamically driven M26I DJ‐1 instability at physiological temperature resolves conflicting literature reports about the behavior of this disease‐associated mutant and illustrates the pitfalls of characterizing proteins exclusively at room temperature or below, as key aspects of their behavior may not be apparent.     

DJ‐1 deficiency triggers microglia sensitivity to dopamine toward a pro‐inflammatory phenotype that is attenuated by rasagiline

DJ‐1 is an oxidative stress sensor that localizes to the mitochondria when the cell is exposed to oxidative stress. DJ‐1 mutations that result in gene deficiency are linked to increased risk of Parkinson's disease (PD). Activation of microglial stress conditions that are linked to PD may result in neuronal death. We postulated that DJ‐1 deficiency may increase microglial neurotoxicity. We found that down‐regulation of DJ‐1 in microglia using an shRNA approach increased cell sensitivity to dopamine as measured by secreted pro‐inflammatory cytokines such as IL‐1β and IL‐6. Furthermore, we discovered that DJ‐1‐deficient microglia had increased monoamine oxidase activity that resulted in elevation of intracellular reactive oxygen species and nitric oxide leading to increased dopaminergic neurotoxicity. Rasagaline, a monoamine oxidase inhibitor approved for treatment of PD, reduced the microglial pro‐inflammatory phenotype and significantly reduced neurotoxicity. Moreover, we discovered that DJ‐1‐deficient microglia have reduced expression of triggering receptor expressed on myeloid cells 2 (TREM2), previously suggested as a risk factor for pro‐inflammation in neurodegenerative diseases. Further studies of DJ‐1‐mediated cellular pathways in microglia may contribute useful insights into the development of PD providing future avenues for therapeutic intervention.

     

Differential regulation of NMDA receptor function by DJ‐1 and PINK1

Dysfunction of PTEN‐induced kinase 1 (PINK1) or DJ‐1 promotes neuronal death and is implicated in the pathogenesis of Parkinson’s disease, but the underlying mechanisms remain unclear. Given the roles of N‐methyl‐d‐ aspartate receptor (NMDAr)‐mediated neurotoxicity in various brain disorders including cerebral ischemia and neurodegenerative diseases, we investigated the effects of PINK1 and DJ‐1 on NMDAr function. Using protein overexpression and knockdown approaches, we showed that PINK1 increased NMDAr‐mediated whole‐cell currents by enhancing the function of NR2A‐containing NMDAr subtype (NR2ACNR). However, DJ‐1 decreased NMDAr‐mediated currents, which was mediated through the inhibition of both NR2ACNR and NR2B‐containing NMDAr subtype (NR2BCNR). We revealed that the knockdown of DJ‐1 enhanced PTEN expression, which not only potentiated NR2BCNR function but also increased PINK1 expression that led to NR2ACNR potentiation. These results indicate that NMDAr function is differentially regulated by DJ‐1‐dependent signal pathways DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR. Our results further showed that the suppression of DJ‐1, while promoted NMDA‐induced neuronal death through the overactivation of PTEN/NR2BCNR‐dependent cell death pathway, induced a neuroprotective effect to counteract DJ‐1 dysfunction‐mediated neuronal death signaling through activating PTEN/PINK1/NR2ACNR cell survival–promoting pathway. Thus, PINK1 acts with DJ‐1 in a common pathway to regulate NMDAr‐mediated neuronal death. This study suggests that the DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR pathways may represent potential therapeutic targets for the development of neuroprotection strategy in the treatment of brain injuries and neurodegenerative diseases such as Parkinson’s disease.     

Loss of DJ‐1 protein stability and cytoprotective function by Parkinson's disease‐associated proline‐158 deletion

DJ‐1 is a ubiquitous protein regulating cellular viability. Recessive mutations in the PARK7/DJ‐1 gene are linked to Parkinson's disease (PD). Although the most dramatic L166P point mutation practically eliminates DJ‐1 protein and function, the effects of other PD‐linked mutations are subtler. Here, we investigated two recently described PD‐associated DJ‐1 point mutations, the A179T substitution and the P158Δ in‐frame deletion. [A179T]DJ‐1 protein was as stable as wild‐type [wt]DJ‐1, but the P158Δ mutant protein was less stable. In accord with the notion that dimer formation is essential for DJ‐1 protein stability, [P158Δ]DJ‐1 was impaired in dimer formation. Similar to our previous findings for [M26I]DJ‐1, [P158Δ]DJ‐1 bound aberrantly to apoptosis signal‐regulating kinase 1. Thus, the PD‐associated P158Δ mutation destabilizes DJ‐1 protein and function. As there is also evidence for an involvement of DJ‐1 in multiple system atrophy, a PD‐related α‐synucleinopathy characterized by oligodendroglial cytoplasmic inclusions, we studied an oligodendroglial cell line stably expressing α‐synuclein. α‐Synuclein aggregate dependent microtubule retraction upon co‐transfection with tubulin polymerization‐promoting protein p25α was ameliorated by [wt]DJ‐1. In contrast, DJ‐1 mutants including P158Δ failed to protect in this system, where we found evidence of apoptosis signal‐regulating kinase 1 (ASK1) involvement. In conclusion, the P158Δ point mutation may contribute to neurodegeneration by protein destabilization and hence loss of DJ‐1 function.     

DJ‐1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle‐like Cells in Response to Sphingosylphosphorylcholine

Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox‐sensitive molecules including a multifunctional protein DJ‐1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)‐stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α‐smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC‐stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ‐1 in hMSCs in response to SPC. The increased abundance of oxidized DJ‐1 in SPC‐stimulated hMSCs was validated by immunoblot analysis. The SPC‐induced increase in the expression of α‐SMA was stronger in DJ‐1‐knockdown hMSCs than in control cells. Moreover, the expression of α‐SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ‐1‐overexpressing hMSCs. Exogenous H₂O₂ mimicked the responses induced by SPC treatment. These results indicate that the ROS‐related DJ‐1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Characteristics of stork feces‐derived H5N1 viruses that are preferentially transmitted to primary human airway epithelial cells

Avian influenza viruses are a possible threat to human health as they may cause an influenza pandemic. Asian open‐bill storks are migratory birds that brought H5N1 viruses into Thailand during the 2004–2005 epidemic. However, to date, there are no reports of direct transmission of stork‐derived H5N1 viruses to Thais. Therefore, we questioned whether or not H5N1 viruses secreted in the feces of infected storks could directly infect cells derived from the human respiratory tract. To answer this question, we used primary NHBE cells as a model. We found that H5N1 viruses from two of the three cloacal swabs rapidly replicated and caused severe structural damage to the infected NHBE cells within the early phase of infection. Viruses from the remaining swab replicated poorly and caused no damage to the infected cells. The rapid‐replicating viruses were able to replicate efficiently even in the presence of a high level of type I IFN production and stimulated a high level of IL‐6 production but not the immunosuppressive cytokine, IL‐10. The genotypic study revealed that the major genotypes of the two rapid‐replicating viruses present in stork feces were the best‐fit genotypes for replication in the primary NHBE cells. In contrast, the major NA‐based genotype found in the cloacal swab containing slow‐replicating viruses could not survive in the primary NHBE cells. Altogether, the data suggested that those stork‐derived H5N1 viruses that preferentially replicated in human airway epithelial cells may exist in nature, and may not require additional mutations in order to defeat the species barrier.     

Structural characterization of a neurotoxic threonine‐rich peptide corresponding to the human prion protein α2‐helical 180–195 segment,and comparison with full‐length α2‐helix‐derived peptides

The 173–195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ‘spots’ of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation‐prone conformations. Here, we report CD and NMR studies on the α2‐helix‐derived peptide of maximal length (hPrP[180–195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other α2‐helix‐derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C‐terminal sequence of the PrP^C full‐length α2‐helix and includes the highly conserved threonine‐rich 188–195 segment. At neutral pH, its conformation is dominated by β‐type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of α‐helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173–179 segment, as occurring in wild‐type and mutant peptides corresponding to the full‐length α2‐helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180–195 parental region in hPrP^C makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full‐length α2‐helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.