The Role of Intrinsically Unstructured Proteins in Neurodegenerative Diseases

The number and importance of intrinsically disordered proteins (IUP), known to be involved in various human disorders, are growing rapidly. To test for the generalized implications of intrinsic disorders in proteins involved in Neurodegenerative diseases, disorder prediction tools have been applied to three datasets comprising of proteins involved in Huntington Disease (HD), Parkinson's disease (PD), Alzheimer's disease (AD). Results show, in general, proteins in disease datasets possess significantly enhanced intrinsic unstructuredness. Most of these disordered proteins in the disease datasets are found to be involved in neuronal activities, signal transduction, apoptosis, intracellular traffic, cell differentiation etc. Also these proteins are found to have more number of interactors and hence as the proportion of disorderedness (i.e., the length of the unfolded stretch) increased, the size of the interaction network simultaneously increased. All these observations reflect that, “Moonlighting” i.e. the contextual acquisition of different structural conformations (transient), eventually may allow these disordered proteins to act as network “hubs” and thus they may have crucial influences in the pathogenecity of neurodegenerative diseases

Identification of HYPK-Interacting Proteins Reveals Involvement of HYPK in Regulating Cell Growth, Cell Cycle, Unfolded Protein Response and Cell Death

<div><p>Huntingtin Yeast Two-Hybrid Protein K (HYPK) is an intrinsically unstructured huntingtin (HTT)-interacting protein with chaperone-like activity. To obtain more information about the function(s) of the protein, we identified 27 novel interacting partners of HYPK by pull-down assay coupled with mass spectrometry and, further, 9 proteins were identified by co-localization and co-immunoprecipitation (co-IP) assays. In neuronal cells, (EEF1A1 and HSPA1A), (HTT and LMNB2) and (TP53 and RELA) were identified in complex with HYPK in different experiments. Various Gene Ontology (GO) terms for biological processes, like protein folding (GO: 0006457), response to unfolded protein (GO: 0006986), cell cycle arrest (GO: 0007050), anti-apoptosis (GO: 0006916) and regulation of transcription (GO: 0006355) were significantly enriched with the HYPK-interacting proteins. Cell growth and the ability to refold heat-denatured reporter luciferase were decreased, but cytotoxicity was increased in neuronal cells where <em>HYPK</em> was knocked-down using <em>HYPK</em> antisense DNA construct. The proportion of cells in different phases of cell cycle was also altered in cells with reduced levels of HYPK. These results show that HYPK is involved in several biological processes, possibly through interaction with its partners.</p> </div

Effect of HYPK on recovery of heat denatured luciferase activity.

<p>The <i>in vivo</i> chaperone activities of Neuro2A-U61 and Neuro2A HYPK-U61 cells in presence and absence of HYPK (before administration of heat shock) are compared (<b><i>A</i></b>). As mentioned in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051415#s2" target="_blank"><b><i>Materials and Methods</i></b></a> section, the cells were subjected to heat shock at 43°C for 1 h (HS) and re-incubated at 37°C CO₂ incubator for the next 6h (HS+R). In every case, heat shock was administered 24h post-transfection of Tet⁺Luc+ve cells with experimental constructs. Comparison of luciferase activities in Neuro2A-U61 cells in presence and absence of HYPK-DsRed in No HS, immediately after heat shock (HS) and HS+R conditions (<b>B</b>). These luciferase activities were also compared upon overexpression of HSPA8-DsRed and 3 other HYPK partners in Neuro2A-U61 cells (<b><i>B</i></b>). Such drop and recovery of the luciferase activities upon heat treatment of HYPK downregulated Neuro2A (Neuro2A-HYPK U61) cells and effect of HYPK and its interacting partners are shown (<b><i>C</i></b>). The ‘n’ and ‘p’ values for Student’s two-tailed t test are indicated in the bar diagrams along with the mean and standard deviation.</p

Squared values of the Pearson Correlation coefficient (R_p) for determination of co-localization of HYPK with its interacting partners.

<p>Extent of co-localization of HYPK with 14 interacting proteins as obtained from confocal imaging studies. The R² values were analyzed to validate whether the co-localization with HYPK was significant. Values greater than or equal to 0.5 were considered to be significant for these co-localization studies.</p

Distribution of cell populations in different cell cycle phases in presence and absence of HYPK in ST<i>Hdh^Q7</i>^/<i>Hdh^Q7</i> cell lines.

<p>Percentage of cell population in different phases of cell cycle (as analyzed by CellQuest Pro software) in presence and absence of HYPK as observed with ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i>cell lines.</p

Preparation of HYPK knock-down neuronal cell lines using the antisense construct (HYPK-U61).

<p>Validation of HYPK knock-down in mRNA and protein levels in Neuro2A (<b><i>A</i></b>) and ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> (<b><i>B</i></b>) cell lines. Expression of both mRNA and protein levels were measured by RT-PCR or SDS-PAGE western analysis in these HYPK knocked-down stable mouse cell lines. Upon exogenous expression of 83Q-DsRed in a dose dependent manner (100 ng and 200 ng), the percentage of cells containing mutant HTT aggregates in presence and absence of endogenous HYPK in ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> cell lines are compared (<b><i>C</i></b>). The ‘n’ and ‘p’ values for Student’s two-tailed t test are indicated in the bar diagrams along with the mean and standard deviation.</p

Cell growth and cell survival in presence and absence of HYPK.

<p>Comparison of the growth curves of control and HYPK downregulated Neuro2A (<b><i>A</i></b>) and ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> (<b><i>B</i></b>) cell lines and effect of exogenous expression of HYPK-DsRed and HSPA8-DsRed in these HYPK knocked-down neuronal cell lines. Comparison of BrdU incorporation in ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> cells overexpressing DsRed and HYPK-DsRed in a dose dependent manner (transfection with 300 ng and 500 ng of plasmid) (<b>C</b>). The difference in BrdU incorporation in ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> cells in absence of HYPK and in presence of HYPK as well as HSPA8 (500 ng) are shown (<b><i>D</i></b>). The effect of mutant Htt (83Q-DsRed) on cell survival of control and HYPK downregulated ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> cells and its effect in presence of exogenous addition of HYPK-DsRed in these cells are shown (<b><i>E</i></b>). Flow cytometry analysis showing distribution of ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> cells in different phases of cell cycle (upon 7-AAD staining) in presence and absence of HYPK (<b>F</b>). The cell cycle analysis was performed using CellQuest Pro software as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051415#s2" target="_blank"><b><i>Materials and Methods</i></b></a> section.The ‘n’ and ‘p’ values for Student’s two-tailed t test are indicated in the bar diagrams along with the mean and standard deviation. The distributions of cell population in different phases are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051415#pone-0051415-t003" target="_blank">Table 3</a>.</p

Formation of high molecular weight HYPK-complex.

<p>High molecular weight HYPK-complex formation by endogenous HYPK with EEF1A1 (<b><i>AI</i></b>) and HSPA1A/HSP70 (<b><i>AII</i></b>) in Neuro2A cells using native-PAGE western analysis; results obtained by similar experiments in ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> and ST<i>Hdh^Q111</i>/<i>Hdh^Q111</i> with LMNB2 (<b><i>BI</i></b>) and HTT (<b><i>BII</i></b>); result obtained by re-probing the blot shown in <b><i>B</i></b> with anti-HYPK antibody is shown in <b><i>BIII</i></b>, showing the difference in migration of purified HYPK and complex-bound HYPK; similar co-IP experiments in ST<i>Hdh^Q7</i>/<i>Hdh^Q7</i> (<b><i>C</i></b>) along with Ponceau staining of the nitrocellulose membrane (<b><i>CI</i></b>). The blot is probed with anti-TP53 (<b><i>CII</i></b>) and anti-RELA (<b><i>CIII</i></b>) antibodies. In all the cases, anti-HYPK antibody was used to pull-down and the immunoprecipitate elute was analyzed to detect the interacting proteins. The loading wells in <b><i>A</i></b> and <b><i>C</i></b> are marked by arrows.</p

HYPK-interacting proteins identified in the present study and obtained earlier.

<p>HYPK-interacting proteins identified by us from pull-down of purified 6HN-HYPK protein as bait with various mammalian SCL as prey (no. 2–28), the HTT-interacting proteins as HYPK partners (no. 29–37) and previously reported HYPK-interacting partners (no. 1 and 38–49).</p