John D. Hulleman

Mechanisms of DJ-1 neuroprotection in a cellular model of Parkinson’s disease

Mitochondrial dysfunction, proteasome inhibition, and α‐synuclein aggregation are thought to play important roles in the pathogenesis of Parkinson’s disease (PD). Rare cases of early‐onset PD have been linked to mutations in the gene encoding DJ‐1, a protein with antioxidant and chaperone functions. In this study, we examined whether DJ‐1 protects against various stresses involved in PD, and we investigated the underlying mechanisms. Expression of wild‐type DJ‐1 rescued primary dopaminergic neurons from toxicity elicited by rotenone, proteasome inhibitors, and mutant α‐synuclein. Neurons with reduced levels of endogenous DJ‐1 were sensitized to each of these insults, and DJ‐1 mutants involved in familial PD exhibited decreased neuroprotective activity. DJ‐1 alleviated rotenone toxicity by up‐regulating total intracellular glutathione. In contrast, inhibition of α‐synuclein toxicity by DJ‐1 correlated with up‐regulation of the stress‐inducible form of Hsp70. RNA interference studies revealed that this increase in Hsp70 levels was necessary for DJ‐1‐mediated suppression of α‐synuclein aggregation, but not toxicity. Our findings suggest that DJ‐1 acts as a versatile pro‐survival factor in dopaminergic neurons, activating different protective mechanisms in response to a diverse range of PD‐related insults.

Site-specific modification of Alzheimer's peptides by cholesterol oxidation products enhances aggregation energetics and neurotoxicity

Accumulation of amyloid β-peptide (Aβ) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimers disease. Schiff-base formation between Aβ and the aldehyde-bearing cholesterol oxidation product 3-β-hydroxy-5-oxo-5,6-secocholestan-6-al is known to increase Aβ amyloidogenicity. Here, we synthesized Aβ variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified Aβ formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from Aβ Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified Aβ under identical conditions and at the same concentration. Our results show that Aβ modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of Aβ.

Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain

Significance Light-chain amyloidosis (AL) is a devastating human disease involving the clonal expansion of a plasma cell and the secretion of destabilized, amyloidogenic immunoglobulin light chains (LCs). Secreted amyloidogenic LCs aggregate extracellularly, leading to proteotoxicity on distal tissues. Available therapeutic strategies to treat AL specifically target the cancerous plasma cell population. While this approach is effective in ∼70% of patients, patients who present with substantial LC-related organ proteotoxicity are generally too sick to tolerate standard chemotherapeutics. Here, we show that stress-independent activation of unfolded protein response-associated transcription factors selectively reduces secretion of amyloidogenic LCs and decreases extracellular soluble LC aggregates associated with proteotoxicity in AL. These results identify a promising therapeutic strategy to treat AL patients unserved by current treatments. Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation.

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson's Disease-Related Protein, DJ-1

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinsons disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the “2O ” form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H2O2 resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a proteins ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.

Translational attenuation differentially alters the fate of disease-associated fibulin proteins

Mutations in fibulin proteins that cause cellular secretion deficiencies are linked to a variety of diseases, ranging from retinopathies to cutis laxa (CL). One secretion‐deficient fibulin mutant, R345W fibulin‐3, causes the macular dystrophy malattia leventinese by increased endoplasmic reticulum retention and/or extracellular misfolding. Herein, we report that small‐molecule activation of the PERK arm of the unfolded protein response partially rescues R345W secretion deficiencies through translational attenuation mediated by eIF2α phosphorylation. Enhanced mutant fibulin‐3 secretion can also be achieved by activation of a PERK‐independent eIF2α kinase through arsenite treatment and is independent of activating transcription factor 4 signaling and protein translation. However, this translational attenuation strategy was unsuccessful for enhancing the secretion deficiencies of fibulin‐5 mutants associated with age‐related macular degeneration or CL. While lowered growth temperature enhanced the secretion of mutants associated with CL (C217R and S227P), these effects were not mediated through translational attenuation. In stark contrast to the situation with fibulin‐3, protein translation was required for efficient wild‐type and mutant fibulin‐5 secretion. These data suggest that alteration of specific cellular signaling pathways and proteostasis network components can differentially influence fibulin fate, a hypothesis that could be exploited as a therapy for fibulin‐related diseases.—Hulleman, J. D., Balch, W. E., Kelly, J. W. Translational attenuation differentially alters the fate of disease‐associated fibulin proteins. FASEB J. 26, 4548–4560 (2012). www.fasebj.org

Compromised mutant EFEMP1 secretion associated with macular dystrophy remedied by proteostasis network alteration

R345W EFEMP1 is secreted poorly, causing the macular dystrophy malattia leventinese. A novel assay shows that other substitutions (F, Y, P) at residue 345 impair secretion, partly by reducing native disulfide bonds. EFEMP1 secretion is rescued by reduced growth temperature and translational attenuation—potential strategies to delay disease.

A High-Throughput Cell-Based Gaussia Luciferase Reporter Assay for Identifying Modulators of Fibulin-3 Secretion

An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3–related diseases.

Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration

An R345W mutation in the N‐glycoprotein, fibulin‐3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N‐Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N‐glycosylation alters the protein homeostasis or proteostasis of wild‐type (WT) and R345W F3 in ARPE‐19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N‐glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N‐glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose‐regulated protein 78) and GRP94 (glucose‐regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N‐glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variants unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N‐glycosylation to acquire a stable, native‐like structure.—Hulleman, J. D., Kelly, J. W. Genetic ablation of N‐linked glycosylation reveals two key folding pathways for R345W fibulin‐3, a secreted protein associated with retinal degeneration. FASEB J. 29, 565‐575 (2015). www.fasebj.org

Alpha-synuclein as a template for the synthesis of metallic nanowires

Exploiting the concepts learnt from nature to build new nanomaterials from the bottom up is critical for the efficient design of complex nanodevices. We demonstrate for the first time that the capacity of the α-synuclein protein to assemble into nanofibres can be used for the synthesis of metallic nanowires. Silver and platinum nanowires with controlled diameters, ranging from 15 to 125 nm, have been synthesized on an α-synuclein protein fibre scaffold.

Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism

Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer’s disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP), were all secreted as efficiently from immortalized human cells as their wild-type (WT) counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively). Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER)-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as ‘site 2’ cleavage). However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K) mutation), A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 (‘site 1’) or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease inhibition function of Cys C.