Meenakshisundaram Balasubramaniam

Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes

Age‐dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP‐43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation‐prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc‐54p/Q40::YFP, a model of polyglutamine array diseases such as Huntingtons, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody‐coupled magnetic beads, were characterized by high‐resolution mass spectrometry. Three Q40::YFP‐associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimers disease models, nematodes with muscle or pan‐neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine‐rich, favoring hydrophobic interactions with other random‐coil domains. A particularly potent modulator of aggregation, CRAM‐1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72‒86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram‐1 reduced β‐amyloid 60% (P < 0.002) and slowed age‐dependent paralysis > 30% (P < 10−6). In wild‐type worms, cram‐1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM‐1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM‐1 (or human orthologs) with ubiquitin, and we verified that CRAM‐1 reduces degradation of a tagged‐ubiquitin reporter. We propose that CRAM‐1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.

Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls

Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease‐specific ‘seed’ proteins; however, roles of other co‐aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimers and control‐subject pools using magnetic‐bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high‐resolution proteomics. Although total detergent‐insoluble aggregates from Alzheimers and controls had similar protein content, within the fractions isolated by tau or Aβ1–42 pulldown, the protein constituents of Alzheimer‐derived aggregates were more abundant, diverse, and post‐translationally modified than those from controls. Tau‐ and Aβ‐containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer‐specific protein enrichment in tau‐containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co‐aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co‐localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer‐specific enrichment in tau‐containing aggregates, were assessed for aggregation‐promoting roles in C. elegans by RNA‐interference ‘knockdown’. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ1–42. These observations imply shared mechanisms driving both types of aggregation, and/or aggregate‐mediated cross‐talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.

Apolipoprotein E4 inhibits autophagy gene products through direct, specific binding to CLEAR motifs

Alzheimer apolipoprotein E (APOE) ɛ4/ɛ4 carriers have earlier disease onset and more protein aggregates than patients with other APOE genotypes. Autophagy opposes aggregation, and important autophagy genes are coordinately regulated by transcription factor EB (TFEB) binding to “coordinated lysosomal expression and regulation” (CLEAR) DNA motifs.

Biological evaluation of 2-arylidene-4, 7-dimethyl indan-1-one (FXY-1): a novel Akt inhibitor with potent activity in lung cancer

AbstractPurpose Human lung cancer is contributed to be a major mortality factor in the cancer-related deaths. Arylidene indan-ones constitute a new class of potential anti-tumor compounds. Herein, we report the biological efficacy of the 2-arylidene-4, 7-dimethyl indan-1-one (FXY-1), a potential lead molecule of arylidene indan-ones in lung cancer models. We previously described anticancer activity of FXY-1 against human breast adenocarcinoma.MethodsFXY-1 efficacy was assessed by standard anticancer screening in NCI-H 460, A549, NCI-H 1975 and NCI-H 2170 cells. Initial molecular docking analysis was performed to check the interaction of compound to Akt enzyme. Anti-proliferation, anti-metastatic and transendothelial cell migration were performed to check efficacy of the drug. Western blot analysis was performed to understand the regulation of pro-apoptotic and anti-apoptotic proteins by the compound. The effect of FXY-1 on caspase induction and Akt phosphorylation were checked using Western blot analysis. Flow cytometry was used to reveal the cell cycle changes and apoptosis-inducing properties of FXY-1 in the lung cancer cells. In-vitro Akt inhibition property of the compound was studied using a fluorescence-based, coupled-enzyme reaction. The in-vivo efficacy of the compound was determined using a mouse xenograft model.ResultsOur molecular docking analysis reveals higher interaction of FXY-1 with Akt. FXY-1 depicted anti-proliferative and pro-apoptotic activity with higher therapeutic window in-vitro in NCI-H 460 and A549 cells. The compound treatment to lung cancer cells resulted in induction of DNA fragmentation, inhibition of transendothelial migration, caspase activation and poly (ADP-ribose) polymerase (PARP) cleavage. FXY-1 treatment resulted in G0/G1 arrest in both cell lines at lower concentrations, but led to apoptosis at higher doses. Western blot analysis revealed dephosphorylation of Akt (Ser 473) with activation of p53, Bax, Bak, Bid and reduction in Bcl-2 and Bcl-xL levels. Further mechanistic investigation showed that FXY-1 activity was facilitated through an allosteric inhibition of Akt enzyme. FXY-1 treatment significantly reduced the tumor growth and pAkt levels in mouse xenograft exhibiting the in-vivo efficacy in the compound.ConclusionCollectively, our results suggest DNA damage-mediated activation by FXY-1 in lung cancer cells leading to extensive apoptosis through the mitochondrial pathway.

Proteins that accumulate with age in human skeletal-muscle aggregates contribute to declines in muscle mass and function in Caenorhabditis elegans

Protein aggregation increases with age in normal tissues, and with pathology and age in Alzheimers hippocampus and mouse cardiac muscle. We now ask whether human skeletal muscle accumulates aggregates with age. Detergent-insoluble protein aggregates were isolated from vastus lateralis biopsies from 5 young (23–27 years of age) and 5 older (64–80 years) adults. Aggregates, quantified after gel electrophoresis, contain 2.1-fold more protein (P<0.0001) when isolated from older subjects relative to young. Of 515 proteins identified by liquid chromatography coupled to tandem mass spectrometry, 56 (11%) were significantly more abundant in older muscle, while 21 (4%) were depleted with age (each P<0.05). Orthologs to seven of these proteins were then targeted in C. elegans by RNA interference. Six of the seven knockdown treatments decreased protein aggregation (range 6–45%, P<0.01 to <0.0001) and increased muscle mass (range 1.5- to 1.85-fold, P<0.01 to <0.0001) in aged nematodes, and rescued mobility (range 1.4 to 1.65-fold, P≤0.0005 each) in a nematode amyloidopathy model. We conclude that specific aggregate proteins, discovered as differentially abundant in aging human muscle, have orthologs that contribute functionally to aggregation and age-associated muscle loss in nematodes, and thus can be considered potential drug targets for sarcopenia in humans.

Structural insights into pro-aggregation effects of C. elegans CRAM-1 and its human ortholog SERF2

Toxic protein aggregates are key features of progressive neurodegenerative diseases. In addition to “seed” proteins diagnostic for each neuropathy (e.g., Aβ1–42 and tau in Alzheimer’s disease), aggregates contain numerous other proteins, many of which are common to aggregates from diverse diseases. We reported that CRAM-1, discovered in insoluble aggregates of C. elegans expressing Q40::YFP, blocks proteasomal degradation of ubiquitinated proteins and thus promotes aggregation. We now show that CRAM-1 contains three α-helical segments forming a UBA-like domain, structurally similar to those of mammalian adaptor proteins (e.g. RAD23, SQSTM1/p62) that shuttle ubiquitinated cargos to proteasomes or autophagosomes for degradation. Molecular modeling indicates that CRAM-1, through this UBA-like domain, can form tight complexes with mono- and di-ubiquitin and may thus prevent tagged proteins from interacting with adaptor/shuttle proteins required for degradation. A human ortholog of CRAM-1, SERF2 (also largely disordered), promotes aggregation in SH-SY5Y-APPSw human neuroblastoma cells, since SERF2 knockdown protects these cells from amyloid formation. Atomistic molecular-dynamic simulations predict spontaneous unfolding of SERF2, and computational large-scale protein-protein interactions predict its stable binding to ubiquitins. SERF2 is also predicted to bind to most proteins screened at random, although with lower average stability than to ubiquitins, suggesting roles in aggregation initiation and/or progression.

ALS-causing mutations in profilin-1 alter its conformational dynamics: A computational approach to explain propensity for aggregation

Profilin-1 (PFN1) is a 140-amino-acid protein with two distinct binding sites―one for actin and one for poly-L-proline (PLP). The best-described function of PFN1 is to catalyze actin elongation and polymerization. Thus far, eight DNA mutations in the PFN1 gene encoding the PFN1 protein are associated with human amyotrophic lateral sclerosis (ALS). We and others recently showed that two of these mutations (Gly118Val or G118V and Cys71Gly or C71G) cause ALS in rodents. In vitro studies suggested that Met114Thr and Thr109Met cause the protein to behave abnormally and cause neurotoxicity. The mechanism by which a single amino acid change in human PFN1 causes the degeneration of motor neurons is not known. In this study, we investigated the structural perturbations of PFN1 caused by each ALS-associated mutation. We used molecular dynamics simulations to assess how these mutations alter the secondary and tertiary structures of human PFN1. Herein, we present our in silico data and analysis on the effect of G118V and T109M mutations on PFN1 and its interactions with actin and PLP. The substitution of valine for glycine reduces the conformational flexibility of the loop region between the α-helix and β-strand and enhances the hydrophobicity of the region. Our in silico analysis of T109M indicates that this mutation alters the shape of the PLP-binding site and reduces the flexibility of this site. Simulation studies of PFN1 in its wild type (WT) and mutant forms (both G118V and T109M mutants) revealed differential fluctuation patterns and the formation of salt bridges and hydrogen bonds between critical residues that may shed light on differences between WT and mutant PFN1. In particular, we hypothesize that the flexibility of the actin- and PLP-binding sites in WT PFN1 may allow the protein to adopt slightly different conformations in its free and bound forms. These findings provide new insights into how each of these mutations in PFN1 might increase its propensity for misfolding and aggregation, leading to its dysfunction.

MMB triazole analogs are potent NF-κB inhibitors and anti-cancer agents against both hematological and solid tumor cells

Triazole derivatives of melampomagnolide B (MMB) have been synthesized via click chemistry methodologies and screened against a panel of 60 human cancer cell lines. Several derivatives showed promising anti-cancer activity, affording growth inhibition (GI50) values in the nanomolar range (GI50 = 0.02-0.99 μM). Lead compound 7h exhibited EC50 values of 400 nM and 700 nM, respectively, against two AML clinical specimens. Compound 7h was significantly more potent than parthenolide as an inhibitor of p65 phosphorylation in both hematological and solid tumor cell lines, indicating its ability to inhibit the NF-κB pathway. In TMD-231 breast cancer cells, treatment with 7h reduced DNA binding activity of NF-κB through inhibition of IKK-β mediated p65 phosphorylation and caused elevation of basal IκBα levels through inhibition of constitutive IκBα turnover and NF-κB activation. Molecular docking and dynamic modeling studies indicated that 7h interacts with the kinase domain of the monomeric IKKβ subunit, leading to inhibition of IKKβ activation, and compromising phosphorylation of downstream targets of the NF-κB pathway; dynamic modeling studies show that this interaction also causes unwinding of the α-helix of the NEMO binding site on IKKβ. Molecular docking studies with 10, a water-soluble analog of 7h, demonstrate that this analog interacts with the dimerization/oligomerization domain of monomeric IKKβ and may inhibit oligomer formation and subsequent autophosphorylation. Sesquiterpene lactones 7h and 10 are considered ideal candidates for potential clinical development.

IL-1β INFLUENCES AUTOPHAGY BY MEDIATING UPREGULATION OF PARKIN AND PARKIN NEDDYLATION IN CELL CULTURE AND ANIMAL MODELS, AND MIMICS THE PATTERN SEEN IN AD BRAIN

results were compared to those of 20 healthy sex and age matched healthy controls(HC). Mechanisms regulating inflammasome signaling involves the priming signal to upregulate the expression of NLRP3 and signal 2 to activate the functional NLRP3; thus monocytes were either primed with LPS(1mg/ml for 2h) and stimulated with Aß42 AlexaFluor488 (FAM)-labeled (10mg/ml) for 24h or stimulated with Aß42 FAM alone for 24h in the presence/absence of Stavudine (50mM) for 22h. Aß-phagocytosis was analyzed by Imagines–FlowCitometry; caspase1 and cytokines were quantified by ELISA. Results: Results showed that: 1) Caspase1, IL1ß and IL18 production by LPS and Aß42 stimulated monocytes of AD patients was significantly increased compared to HC (for all p<0.05); 2) Aß-phagocytosis was significantly reduced in LPS-primed and Aß42 stimulated cells of AD and HC individuals compared to those stimulated with Aß42 alone (for both p<0.05); 3) Stavudine resulted in a drastic reduction of Caspase1, IL1ß and IL18 production (p<0.05) but did not modify the Aß-phagocytosis capacity of monocytes. Conclusions:NLRP3 inflammasome-driven inflammation reduces monocytes mediated Aß phagocytosis in AD and HC. Stavudine dampens NLRP3 activation and downstream inflammation but does not significantly modify Aß-phagocytosis. These results suggest that, even if Stavudine could be useful in modulating neuroinflammation in AD, AD-associated impairments in Aßphagocytosis are not a consequence of a direct consequence of inflammation.

Involvement of tRNAs in replication of human mitochondrial DNA and modifying effects of telomerase

Overexpression of telomerase has been shown to significantly increase the lifespan of mice. When mechanistically attributed to repair of critically short telomeres, the lifespan extending action of telomerase cannot be reconciled with the observation that telomerase-null mice do not exhibit shortening of lifespan for at least two generations. We hypothesized that telomerase may interfere with replication of mitochondrial DNA (mtDNA) in a way that reduces formation of deletions - the primary cause of age-dependent cell attrition in non-renewable cells such as myocytes and neurons. Here we show that several tRNA genes may function as alternative origins of replication (ORIs). We also show that telomerase interacts with canonical light strand ORI (ORIL) and tRNAs and modifies their activities. Our results suggest that replication of mitochondrial DNA (mtDNA) proceeds through a variety of mechanisms resulting in a mixture of classic strand-displacement mode, and coupled replication of heavy and light strands. Our results also suggest that effects of telomerase may arise from binding ORIL and thus limiting contribution of the deletion-prone strand displacement mode to mtDNA synthesis. These findings imply that it may be possible to uncouple detrimental and beneficial effects of telomerase, and thereby to improve telomerase-based strategies to extend lifespan.