The Emerging Role of Proteolysis in Mitochondrial Quality Control and the Etiology of Parkinson's Disease

Mitochondria are highly dynamic organelles that are important for many diverse cellular processes, such as energy metabolism, calcium buffering, and apoptosis. Mitochondrial biology and dysfunction have recently been linked to different types of cancers and neurodegenerative diseases, most notably Parkinson's disease. Thus, a better understanding of the quality control systems that maintain a healthy mitochondrial network can facilitate the development of effective treatments for these diseases. In this perspective, we will discuss recent advances on two mitochondrial quality control pathways: the UPS and mitophagy, highlight how new players may be contributing to regulate these pathways. We believe the proteases involved will be key and novel regulators of mitochondrial quality control, and this knowledge will provide insights into future studies aimed to combat neurodegenerative diseases

Solution structure of the carboxy-terminal Tudor domain from human Coilin

AbstractThe Cajal body is a dynamic eukaryotic nuclear organelle that is known primarily as an organizational center for the assembly of snRNAs involved in transcript splicing. One of the most critical components of the Cajal body is the scaffolding protein, Coilin. Here, we demonstrate by NMR methods that the carboxy-terminal region contains a Tudor domain. The Tudor domain is atypical due to the presence of several unstructured loops, one greater than thirty amino acids in length. Tudor domains have been noted previously to bind DNA, RNA and modified amino acids. The absence of these sequence and structural signatures in the Coilin Tudor domain supporting these established functions suggests an alternative role

Solution Structure and Peptide Binding of the PTB Domain from the AIDA1 Postsynaptic Signaling Scaffolding Protein

<div><p>AIDA1 links persistent chemical signaling events occurring at the neuronal synapse with global changes in gene expression. Consistent with its role as a scaffolding protein, AIDA1 is composed of several protein-protein interaction domains. Here we report the NMR structure of the carboxy terminally located phosphotyrosine binding domain (PTB) that is common to all AIDA1 splice variants. A comprehensive survey of peptides identified a consensus sequence around an NxxY motif that is shared by a number of related neuronal signaling proteins. Using peptide arrays and fluorescence based assays, we determined that the AIDA1 PTB domain binds amyloid protein precursor (APP) in a similar manner to the X11/Mint PTB domain, albeit at reduced affinity (∼10 µM) that may allow AIDA1 to effectively sample APP, as well as other protein partners in a variety of cellular contexts.</p></div

Deubiquitinating enzymes regulate PARK2-mediated mitophagy.

The selective degradation of mitochondria by the process of autophagy, termed mitophagy, is one of the major mechanisms of mitochondrial quality control. The best-studied mitophagy pathway is the one mediated by PINK1 and PARK2/Parkin. From recent studies it has become clear that ubiquitin-ligation plays a pivotal role and most of the focus has been on the role of ubiquitination of mitochondrial proteins in mitophagy. Even though ubiquitination is a reversible process, very little is known about the role of deubiquitinating enzymes (DUBs) in mitophagy. Here, we report that 2 mitochondrial DUBs, USP30 and USP35, regulate PARK2-mediated mitophagy. We show that USP30 and USP35 can delay PARK2-mediated mitophagy using a quantitative mitophagy assay. Furthermore, we show that USP30 delays mitophagy by delaying PARK2 recruitment to the mitochondria during mitophagy. USP35 does not delay PARK2 recruitment, suggesting that it regulates mitophagy through an alternative mechanism. Interestingly, USP35 only associates with polarized mitochondria, and rapidly translocates to the cytosol during CCCP-induced mitophagy. It is clear that PARK2-mediated mitophagy is regulated at many steps in this important quality control pathway. Taken together, these findings demonstrate an important role of mitochondrial-associated DUBs in mitophagy. Because defects in mitochondria quality control are implicated in many neurodegenerative disorders, our study provides clear rationales for the design and development of drugs for the therapeutic treatment of neurodegenerative diseases such as Parkinson and Alzheimer diseases

<i>(a)</i> Sequence alignment of the AIDA1 PTB domain against the APP binding proteins, Dab1 [25], X11 [17] and Fe65 [20].

<p>Five aromatic amino acids selected for alanine substitution in AIDA1 PTB domain are boxed. <i>(b)</i> Backbone atom superposition of top15 structures according to lowest refinement energy. <i>(c)</i> Strip plots of a ¹³C-edited NOESY spectrum at the Cβ chemical shift of each alanine substituted in the PTB5M mutant. A asterisk denotes a resonance not associated with that strip. <i>(d)</i> A ribbon representation of the PTB5M model highlighting the positions of the alanine substitutions. Y6A is not shown in the figure as the first 14 amino acids are unstructured and were excluded from the structure calculation.</p

Affnities of APP-derived peptides for two solubility enhanced mutants of the AIDA1 PTB domain. ND: not done.

<p>Affnities of APP-derived peptides for two solubility enhanced mutants of the AIDA1 PTB domain. ND: not done.</p

Structural similarity of the AIDA1 PTB domain to related PTB domains that also bind APP.

<p>Structural similarity of the AIDA1 PTB domain to related PTB domains that also bind APP.</p

Solubilities and thermal denaturation midpoints of the AIDA PTB domain and alanine substitution mutants.

<p>Solubilities and thermal denaturation midpoints of the AIDA PTB domain and alanine substitution mutants.</p

Amino acid preferences of the AIDA1 PTB domain for APP determined from a peptide array.

<p>A list of peptides on the array are provided in supplementary material. <i>(a)</i> The array probed with anti 6xHis mAb only. Positive control 6xHis peptides are identified by a <b>+</b>. <i>(b)</i> The array probed with 6xHis-AIDA1 PTB domain. <i>(c)</i> Sliding window peptide scan of 12-mers spanning aa. 672–697 of APP. Peptides are duplicated on the array; for example, at A3 and A18. Since peptide content per spot can vary, if a signal was observed at the exposure presented it was deemed to be interaction. <i>(d)</i> Results of a window scan across the APP C-terminal sequence and an exhaustive positional scan. Grey boxes indicate binding was observed, regardless of signal intensity.</p

Titration of FITC-labeled APP peptides with a solubility enhanced mutant (Y70A) of the AIDA1 PTB domain.

<p>Binding was monitored by fluorescence anisotropy. Legend: APP17, a short X11-like binding site; APP32, a longer Fe65-like binding site; APP17{pY}, a short X11-like phosphopeptide. The peptide sequences are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065605#pone-0065605-t004" target="_blank">Table 4</a>.</p