Search

PINK1 phosphorylates ubiquitin, which then binds to Parkin and activates its E3 ligase activity, leading to induction of selective autophagy of damaged mitochondria., PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.

Differential localization to the inner and outer mitochondrial membranes regulates PINK1 stability and function., PINK1 is a mitochondrial kinase mutated in some familial cases of Parkinson’s disease. It has been found to work in the same pathway as the E3 ligase Parkin in the maintenance of flight muscles and dopaminergic neurons in Drosophila melanogaster and to recruit cytosolic Parkin to mitochondria to mediate mitophagy in mammalian cells. Although PINK1 has a predicted mitochondrial import sequence, its cellular and submitochondrial localization remains unclear in part because it is rapidly degraded. In this study, we report that the mitochondrial inner membrane rhomboid protease presenilin-associated rhomboid-like protein (PARL) mediates cleavage of PINK1 dependent on mitochondrial membrane potential. In the absence of PARL, the constitutive degradation of PINK1 is inhibited, stabilizing a 60-kD form inside mitochondria. When mitochondrial membrane potential is dissipated, PINK1 accumulates as a 63-kD full-length form on the outer mitochondrial membrane, where it can recruit Parkin to impaired mitochondria. Thus, differential localization to the inner and outer mitochondrial membranes appears to regulate PINK1 stability and function.

Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including Parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin’s translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3-/- mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin substrates other than VDAC1.

Recent developments in the field of protein separation allows for the analysis of qualitative and quantitative global protein changes in a particular state of a biological system. Due to the enormous number of proteins potentially present in a cell, sub-fractionation and the enrichment of specific organelles are emerging as a necessary step to allow a more comprehensive representation of the protein content. The proteomic studies demonstrate that a key to understand the mechanisms underlying physiological or pathological phenotypes lies, at least in part, in post-translational modifications (PTMs), including phosphorylation of proteins. Rapid improvements in proteomic characterization of amino acid modifications are further expanding our comprehension of the importance of these mechanisms. The present review will provide an overview of technologies available for the study of a proteome, including tools to assess changes in protein quantity (abundance) as well as in quality (PTM forms). Examples of the recent application of these technologies and strategies in the field of kinase signalling will be provided with particular attention on the role of PKC in the heart. Studies of PKC-mediated phosphorylation of cytoskeletal, myofilament and mitochondrial proteins in the heart have provided great insight into the phenotypes of heart failure, hypertrophy and cardioprotection. Proteomics studies of the mitochondria have provided novel evidences for kinase signalling cascades localized to the mitochondria, some of which are known to involve various isoforms of PKC. Proteomics technologies allow for the identification of the different PTM forms of specific proteins and this information is likely to provide insight into the determinants of morphological as well as metabolic mal-adaptations, both in the heart and other tissues.

Keratin 17 (K17) is a type I intermediate filament protein that is constitutively expressed in ectoderm-derived epithelial appendages and robustly induced in epidermis following injury, during inflammation, and in chronic diseases such as psoriasis and cancer. Mutations within K17 are responsible for two rare diseases related to ectodermal dysplasias. Studies in K17-null mice uncovered several roles for K17, including structural support, resistance to TNFα-induced apoptosis, regulation of protein synthesis, and modulation of cytokine expression. Yet, little is known about the regulation of K17 protein via post-translational modification. Here, we report that serine 44 in the N-terminal head domain of K17 (K17-Ser(44)) is phosphorylated in response to extracellular stimuli (serum, EGF, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate) that alter skin keratinocyte growth, and to cellular stresses (sorbitol-induced hyperosmotic shock, UV irradiation, and hydrogen peroxide-induced oxidative stress). It also occurs in basaloid skin tumors in situ. Upon its stimulation in skin keratinocytes, K17-Ser(44) phosphorylation is induced rapidly but stays on transiently. The majority of the phosphorylated K17-Ser(44) pool is polymer-bound and is not obviously related to a change in filament organization. The amino acid sequence surrounding K17-Ser(44) matches the consensus for the AGC family of basophilic kinases. We show that p90 RSK1, an AGC kinase involved in the regulation of cell survival and proliferation, phosphorylates K17-Ser(44) in skin keratinocytes. These findings confirm and expand the tight link that has emerged between K17 up-regulation and growth and stress responses in the skin epithelium.

Polyubiquitin chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyubiquitin signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyubiquitin signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity Lys63 polyubiquitin-binding proteins and demonstrate their specificity in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyubiquitin-linkage dependence of NF-κB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1β and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic and biochemical applications for these tools and extension of the design strategy to other polymeric ubiquitin-like protein modifications.

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n = 3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked site-specific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-nitrosation of mitochondrial permeability transition-associated proteins represents an intriguing potential link to cardioprotection.

Mitochondria are dynamic organelles that usually exist in extensive and interconnected networks that undergo constant remodeling through fission and fusion. These processes are governed by distinct sets of proteins whose mechanism and regulation we are only beginning to fully understand. Early studies on mitochondrial dynamics were performed in yeast and simple mammalian cell culture models that allowed easy visualization of these intricate networks. Equipped with this core understanding, the field is now expanding into more complex systems. Cardiac cells are a particularly interesting example because they have unique energetic and spatial demands that make the study of their mitochondria both challenging and potentially very fruitful. This review will provide an overview of mitochondrial fission and fusion as well as recent developments in the understanding of these processes in the heart.

Background— Cardiac resynchronization therapy (CRT) improves chamber mechanoenergetics and morbidity and mortality of patients manifesting heart failure with ventricular dyssynchrony; however, little is known about the molecular changes underlying CRT benefits. We hypothesized that mitochondria may play an important role because of their involvement in energy production. Methods and Results— Mitochondria isolated from the left ventricle in a canine model of dyssynchronous or resynchronized (CRT) heart failure were analyzed by a classical, gel-based, proteomic approach. Two-dimensional gel electrophoresis revealed that 31 mitochondrial proteins where changed when controlling the false discovery rate at 30%. Key enzymes in anaplerotic pathways, such as pyruvate carboxylation and branched-chain amino acid oxidation, were increased. These concerted changes, along with others, suggested that CRT may increase the pool of Krebs cycle intermediates and fuel oxidative phosphorylation. Nearly 50% of observed changes pertained to subunits of the respiratory chain. ATP synthase-β subunit of complex V was less degraded, and its phosphorylation modulated by CRT was associated with increased formation (2-fold, P =0.004) and specific activity (+20%, P =0.05) of the mature complex. The importance of these modifications was supported by coordinated changes in mitochondrial chaperones and proteases. CRT increased the mitochondrial respiratory control index with tightened coupling when isolated mitochondria were reexposed to substrates for both complex I (glutamate and malate) and complex II (succinate), an effect likely related to ATP synthase subunit modifications and complex quantity and activity. Conclusions— CRT potently affects both the mitochondrial proteome and the performance associated with improved cardiac function.

Rationale : We previously discovered several phosphorylations to the β subunit of the mitochondrial F 1 F o ATP synthase complex in isolated rabbit myocytes on adenosine treatment, an agent that induces cardioprotection. The role of these phosphorylations is unknown. Objective : The present study focuses on the functional consequences of phosphorylation of the ATP synthase complex β subunit by generating nonphosphorylatable and phosphomimetic analogs in a model system, Saccharomyces cerevisiae . Methods and Results : The 4 amino acid residues with homology in yeast (T58, S213, T262, and T318) were studied with respect to growth, complex and supercomplex formation, and enzymatic activity (ATPase rate). The most striking mutant was the T262 site, for which the phosphomimetic (T262E) abolished activity, whereas the nonphosphorylatable strain (T262A) had an ATPase rate equivalent to wild type. Although T262E, like all of the β subunit mutants, was able to form the intact complex (F 1 F o ), this strain lacked a free F 1 component found in wild-type and had a corresponding increase of lower-molecular-weight forms of the protein, indicating an assembly/stability defect. In addition, the ATPase activity was reduced but not abolished with the phosphomimetic mutation at T58, a site that altered the formation/maintenance of dimers of the F 1 F o ATP synthase complex. Conclusions : Taken together, these data show that pseudophosphorylation of specific amino acid residues can have separate and distinctive effects on the F 1 F o ATP synthase complex, suggesting the possibility that several of the phosphorylations observed in the rabbit heart can have structural and functional consequences to the F 1 F o ATP synthase complex.

The ATP synthase complex is a critical enzyme in the energetic pathways of cells because it is the enzyme complex that produces the majority of cellular ATP. It has been shown to be involved in several cardiac phenotypes including heart failure and preconditioning, a cellular protective mechanism. Understanding the regulation of this enzyme is important in understanding the mechanisms behind these important phenomena. Recently there have been several post-translational modifications (PTM) reported for various subunits of this enzyme complex, opening up the possibility of differential regulation by these PTMs. Here we discuss the known PTMs in the heart and other mammalian tissues and their implication to function and regulation of the ATP synthase.

Proteomic analysis of heart tissue is complicated by the large dynamic range of its proteins. The most abundant proteins are the myofilament proteins, which comprise the contractile apparatus. This chapter describes a protocol for fractionation of heart tissue that extracts the myofilament proteins into a separate sample fraction, allowing analysis of lower-abundance proteins. Importantly, this is performed in a manner that is compatible with two-dimensional electrophoresis and high-performance liquid chromatography, two of main technologies of proteomics. The method produces three fractions based on solubility at different pHs: (1) cytoplasmic-enriched extract (neutral pH), (2) myofilament-enriched extract (acidic pH), and (3) membrane protein-enriched pellet. Fractionation of heart tissue in this manner provides the basis for in-depth proteomic analysis.

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.

Ischemic preconditioning is characterized by resistance to ischemia reperfusion injury in response to previous short ischemic episodes, a protective effect that can be mimicked pharmacologically. The underlying mechanism of protection remains controversial and requires greater understanding before it can be fully exploited therapeutically. To investigate the overall effect of preconditioning on the myocardial proteome, isolated rabbit ventricular myocytes were treated with drugs known to induce preconditioning, adenosine or diazoxide (each at 100 μmol/L for 60 minutes). Their protein profiles were then compared with vehicle-treated controls (n=4 animals per treatment) using a multitiered 2D gel electrophoresis approach. Of 28 significantly altered protein spots, 19 nonredundant proteins were identified (5 spots remained unidentified). The majority of these proteins are involved in mitochondrial energetics, including subunits of tricarboxylic acid cycle enzymes and oxidative phosphorylation complexes. These changes were not indiscriminate, with only a small number of enzymes or complex subunits altered, indicating a very specific and targeted affect of these 2 preconditioning mimetics. Among the changes were shifts in the extent of posttranslational modification of 4 proteins. One of these, the adenosine-induced phosphorylation of the ATP synthase β subunit, was fully characterized with the identification of 5 novel phosphorylation sites. This proteomics approach provides an overall assessment of the cellular response to pharmacological treatment with adenosine and diazoxide and identifies a distinct subset of enzymes and protein complex subunit that may underlie the preconditioned phenotype.