p62/sequestosome 1 - p62在蛋白質堆疊物的降解作用之調控機制

Chapter 1. Introduction

1.6 p62/sequestosome 1

The complementary (c)DNA of p62 was cloned in 1996 [147], and the encoding protein was subsequently named sequestosome 1, on account of its ability to form cytosolic inclusion bodies with ubiquitinated proteins, and to ”sequester” these proteins in specialized compartments [148]. p62 messenger (m)RNA is ubiquitously expressed in a variety of tissues [147], and the protein is conserved throughout metazoa. Multiple protein-protein interaction motifs have been characterized in the

p62 protein [149] (Fig 1.2). At the N-terminus of p62, there is a PB1 domain which

mediates self interaction. As such, p62 can bind to other proteins containing PB1;

these proteins are usually signaling molecules, including PKCζ, MEKK3, MEK5 and

ERK, and p62 acts as a scaffold protein to regulate the various signaling pathways [150, 151]. In addition, p62 can assemble into homopolymers via the PB1 domain, which is crucial for the formation of inclusion bodies or sequestosome structures. It is interesting to note that this domain also interacts with the Rpt1 subunit of the proteasome, suggesting a role for p62 in the proteasomal degradative system.

Fig 1.2 Schematic layout of the domain organization of p62

p62 dimerizes with another p62 molecule via binding of the PB1, Phox/Bem1 homology 1 (PB1) domain. This domain also interacts with the Rpt1 subunit of the proteasome. The LC3-interacting region (LIR) of p62 interacts with LC3, and the

ubiquitin associated (UBA) domain binds K63-polyubiquitin chains.

(Adapted from [152])

The C-terminus of p62 contains an ubiquitin-associated (UBA) domain which allows it to bind to ubiquitinated proteins, and in particular K63-polyubiquitinated proteins, with high affinity [153]. It has been reported that this domain enables p62 to co-localize with certain neurodegeneration-related polyubiquitinated protein aggregates, such as α-synuclein, Tau, and Huntingtin (Htt), in brain autopsies from patients [145, 146]. Interestingly, overexpression of p62 mutated in either the PB1 domain or the UBA motif significantly reduced the number of sequestosomes, as compared to those observed in cells expressing wild type p62 [154]. This finding indicates that both p62 self interaction and association of ubiquitin-positive proteins with p62 are required for the formation of inclusion bodies. Of note, the UBA domain is also required to recognize cargo for sorting to the autophagy or proteasome, which will be discussed in later sections.

Other motifs for protein interaction in p62 include a ZZ domain for AMP receptor binding [155], a TBS domain for association with an E3 ligase, TRAF6 [156], and a KIR domain for association with Keap1, a negative regulator of Nrf2 [157]. A recent study also identified two nuclear localization signal domains (NLS1 and NLS2) and one nuclear export motif (NES) in p62 [158]. It also showed that nuclear p62 contributes to nuclear protein aggregate formation in frontotemporal lobar degeneration and spinocerebellar ataxia.

p62 as a shuttle factor for proteasomal degradation

The presence of a UBA domain, a TBS domain (for TRAF6 binding) and a PB1 domain (for direct targeting to the proteasome subunit Rpt1) strongly suggest involvement of p62 in proteasome degradation. In support of this hypothesis, depletion of p62 expression in a cell model system resulted in accumulation of polyubiquitinated proteins [153]. In addition, two substrates, TrkA and Tau, have been reported to be shuttled to the proteasome in a p62-dependent manner. Upon NGF stimulation, the NGF receptor, TrkA, is subject to K63-linked polyubiquitination by TRAF6, and subsequently interacts with the UBA domain of p62. This association targets TrkA to Rpt1 and facilitates deubiquitination of TrkA, which is essential for its turnover by the proteasome [159]. Therefore, depletion of p62 results in stabilization of TrkA in PC12 cells. On the other hand, Tau- and ubiquitin-positive inclusion bodies in AD brains were found to co-localize with the E3 ligase TRAF6 and p62. Depletion of p62 blocked the Tau-proteasome (Rpt1) interaction and Tau degradation [160].

Furthermore, neurofibrillary tangle-like structures containing hyperphosphorylated Tau were observed in the brains of p62^–/– mice [161]. It remains possible that homeostasis of other substrates may also be regulated through the TRAF6/p62/proteasome axis, pending further investigation.

p62 as a cargo receptor in selective autophagy

In addition to its role as a shuttling factor for proteasome degradation, Bjorkoy et al. (2005) demonstrated that some p62 proteins are constrained inside LC3-positive

autophagosomes. Blockage of autophagy increases the size and number of p62-containing inclusion bodies [154], suggesting a role for p62 in autophagic degradation. More recently, an LC3-interacting region (LIR) was identified within p62, N-terminally proximal to its UBA domain. Biochemical analyses showed that the LIR domain is required for p62-LC3 binding and the subsequent entrapment of p62 into autophagosomes for degradation [140, 141]. In cultured cells, overexpression of p62 mutated in either the LIR or UBA domain results in a significant increase in cytosolic ubiquitin-positive inclusion bodies [141], similar to those observed in neurodegenerative diseases. In the first part of my PhD project (described in chapter 2), I identified some of the residues in LC3 essential for LC3-p62 complex formation, and demonstrated the importance of this conserved p62-LC3 interaction in the turnover of cytotoxic Huntingtin aggregates.

It has become clear that the p62-LC3 complex not only mediates aggrephagy, but is also required for selective autophagic degradation of post-mitotic midbody rings, damaged mitochondria (mitophagy), excessive peroxisomes (pexophagy) and intracellular pathogens such as S. enteric (xenophagy) [138]. A recent study found that

during Mycobacterium tuberculosis infection, p62 recruits antibacterial proteins (rather than bacteria) into autolysosomes, which results in bacterial death when the bacteria-containing phagosomes fuse with autolysosomes [162]. In all of the selective autophagy processes described above, ubiquitination is a prerequisite for p62 to recognize specific substrates.

It should be noted that autophagic receptors other than p62 have been recently identified. These proteins, which include NBR1, Nix and NDP52, contain at least one LIR domain that interacts with LC3 [163-165]. These cargo receptors are believed to coordinately regulate substrate selectivity under different conditions, although elucidation of the underlying mechanisms awaits further investigation.

Regulation of p62 expression

Expression of the multifunctional p62 protein requires extensive regulation.

Transcription of p62 has been shown to be rapidly activated by a variety of extracellular signals, including phorbol 12-myristate 13-acetate (PMA) and calcium ionomycin in peripheral blood mononuclear cells, serum and platelet-derived growth factor (PDGF) in serum-starved NIH3T3 cells [147], and prostaglandin J2 (PGJ2) in neuroblastoma SK-N-SH cells [166]. Analysis of the 5’-flanking region of the p62 gene revealed the presence of putative binding sites for the transcription factors AP-1,

Sp1, NF-κB, c-myc, Ets-1 family proteins, MyoD, and C/EBP [167], which may coordinately contribute to the early p62 induction response. It has been reported that the oncogene Ras induces binding of the activator protein (AP)-1 to the p62 promoter, thereby enhancing the production of p62 in human cancer cells [168]. In addition, several of the aforementioned putative transcription factor binding sites are embedded in CpG islands, which are sensitive to oxidative damage [169, 170]. Therefore, the expression of p62 can also be attenuated under conditions of chronic oxidative stress that commonly occur in aging. On the other hand, acute induction of oxidative stress by electrophile treatment also simulates p62 transcription, by activating nuclear factor (NF)-E2-related factor 2 (Nrf2) [171], a redox-sensitive transcription factor, indicating a pivotal role of p62 in response to oxidative and electrophilic stress.

The pathogenic functions of p62 in disease

Altered expression of p62 has been linked to various diseases. An abundance of p62 proteins is linked to breast tumors, liver cancers and liver cirrhosis [172, 173]. A mouse model with reduced levels of a key autophagic molecule, beclin 1, developed spontaneous liver and lung tumors with accumulation of p62 protein [174].

Furthermore, liver tumors developed spontaneously in a liver-specific ATG7-deficient mouse model; this was suppressed by depletion of p62 protein, suggesting that

increased levels of p62 contribute to tumorgenesis [175]. Additional studies have shown that p62 is an important mediator of Ras-induced lung adenocarcinomas. The

interaction between p62 and TRAF6 triggers polyubiquitination of IκB kinase (IKK), which in turn activates NFκB signaling [176], a major signaling pathway downstream

of Ras. Therefore, Ras induces excessive expression of p62, thereby activating the NFκB pathway in cancer cells; this in turn decreases the number of reactive oxygen

species (ROS) and enhances tumor survival [168]. In addition, the KIR sequence of p62 also allows it to chelate Keap1, a negative regulator of the stress responsive transcription factor Nrf2 [157]. Up-regulation of p62 in tumor cells may therefore also alleviate oxidative stress by promoting Nrf2 activity, which in turn induces the expression of key ROS scavengers [177]. As mentioned in previous section, Nrf2 enhances p62 transcription, resulting in a positive feedback loop that prolongs the antioxidative response under conditions of stress.

Mutations of p62 are also implicated in Paget’s disease of bone (PDB), a disease characterized by abnormal bone destruction and regrowth. Most of the mutations are within or surrounding the UBA region, indicating that impaired binding of p62 to ubiquitin contributes to the pathogenesis of PDB [178]. However, the most prevalent PDB mutation, P392L, does not alter the binding affinity of p62 to ubiquitin, and nor does it affect the formation of the sequestosome [179, 180]. Instead, the

P392L mutation reduces interaction between p62 and the tumor suppressor Cyclindromatosis (CYLD), a deubiquitinase enzyme that negatively regulates osteoclastogenesis [181]. The p62-CYLD complex may be the key for osteoclast development/bone resorption activity in PDB.

The presence of p62 in neuronal inclusions of a variety of neurodegenerative diseases implies a crucial role of this protein in neuropathy. The ability of p62 to serve as a scaffold and act as a shuttle factor in the proteasomal and selective autophagic systems supports the hypothesis that its protective function involves sequestering misfolded proteins and mediating their turnover. The case for this hypothesis was strengthened by a recent study demonstrating that p62 immunostaining co-localized with pathological inclusions in PD brains, but was homogenously distributed in healthy neurons of both control and PD brains [182].

Several studies have provided evidence that PD-associated PINK1 and parkin mutations might work together to impair mitophagy and promote the pathogenesis of PD [183, 184]. Depolarization of the mitochondrial membrane triggers accumulation of the mitochondrial kinase PINK1 at the outer mitochondrial membrane, which in turn induces translocation of the E3 ligase parkin to mitochondria. Parkin catalyses K27 and K63-linked ubiquitination of voltage-dependent anion channel 1 (VDAC1) on depolarized mitochondria, allowing binding of p62 and subsequent recruitment of

LC3 to accomplish mitophagy [183, 184]. Therefore, down-regulation of p62 may also contribute to defects in mitochondrial turnover in PD.

The role of p62 in AD has recently come under increased scrutiny. Du et. al.

observed an age-dependent increase in oxidative damage to the p62 promoter in the brains of patients with sporadic AD, and this was inversely correlated with the level of p62 protein [169]. Overall, it seems that p62 functions as a neuroprotective protein in AD, although the regulatory mechanisms of this protein remain to be determined.

The relationship between p62 and AD is discussed in detail in the second part of my PhD project (described in chapter 3). I also address the role of a novel transcriptional control pathway in regulating p62 expression in familial AD.

In summary, we are intrigued by the multifunctional and protective role of p62 in the clearance of aggregation-prone proteins. In the following chapters, we focus on the role of the evolutionarily conserved LC3-p62 interaction in the autophagic removal of neurodegeneration-associated Htt aggregates, and FAD-associated regulation of p62 expression.

Chapter 2 The evolutionarily conserved cargo-receptor binding site of LC3 interacts with p62 to mediate autophagy-dependent degradation of mutant

Huntingtin

2.1 Abstract

Autophagy has been found to play a critical role in the clearance of various aggregated proteins that contribute to the pathogenesis of human diseases. In mammalian cells, p62/sequestosome-1 protein binds to both LC3 and polyubiquitinated cargo proteins destined to autophagy-mediated degradation. LC3, the mammalian homologue of yeast Atg8, is translocated to autophagosomes upon induction of autophagy and regulates autophagosome formation in cultured mammalian cells. Our previous findings have identified several hydrophobic or positively charged residues in Atg8 that are essential for its interaction with the cargo receptor Atg19 in selective autophagic processes of yeast. We thus sought to determine whether such interaction is evolutionally conserved from yeast to mammals.

Using amino acid replacement approach, we demonstrated that cells expressing mutant LC3 (LC3-K30D, LC3-K51A, or LC3-L53A) all exhibit disrupted LC3-p62 interaction and impaired autophagic degradation of p62, suggesting that the p62-binding site of LC3 is localized within an evolutionarily conserved domain.

While cells overexpressing LC3 mutants did not exhibit normal autophagic activity, the autophagy-mediated clearance of aggregation–prone mutant Huntingtin, the cytotoxic protein inducing pathological phenotypes of Huntintung’s disease, was significantly defective in cells expressing LC3 mutants. These results suggest that p62

directly binds to the evolutionarily conserved cargo-receptor binding domain of Atg8/LC3 and selectively mediates the clearance of mutant Huntingtin aggregates.

2.2 Introduction

Autophagy, one of the main degradative pathways in eukaryotic cells, is tightly governed by a series of autophagy-related preoteins (Atgs) [185, 186]. Among them, Atg8 in yeast and its homologue, the microtubule-associated protein (MAP) light chain 3 (LC3), are the most reliable markers of autophagosome. The conversion of cytosolic Atg8/LC3-I to PE-conjugated Atg8-PE/LC3-II is tightly correlated with the numbers of autophagosome and the activation of autophagy [111, 187-189].

Selective autophagy has been better characterized in yeast. Atg8 can interact with a cargo receptor, Atg19, which binds to the precursor of aminopeptidase 1 (prApe1), a vacuolar enzyme [190]. The Atg8-Atg19 interaction is thus necessary for the specific delivery of prApe1 into vacuoles through autophagic transport vesicles (cargo sorting mechanism) and for the formation of mature Ape1 [191]. However, molecules governing the cargo-sorting mechanism of selective autophagy remain elusive in mammals.

A number of studies have indicated that the p62/sequestosome 1 protein may function as a cargo receptor to recruit particular proteins into autophagosomes. When

p62 mutants that lack LC3-binding ability are expressed in p62^-/- cells, increased intracellular accumulations of polyubiquitinated proteins are only colocalized with p62, but not LC3 [141]. Of noted, p62 has been found to colocalize with certain

neurodegeneration-related insoluble aggregates containing polyubiquitinated proteins, such as Tau, Huntingtin (Htt), and α-synuclein [145, 146]. These findings suggest that

p62 could play a crucial role in the clearance of insoluble inclusion bodies by shuttling those aggregation-prone proteins to autophagy through its specific associations with both LC3 and polyubiquitin chain. Therefore, the identification of p62-binding residues within LC3 could delineate the role of the p62-LC3 interaction in selective autophagy-mediated clearance of neurodegeneration-related protein aggregates.

Previous studies have characterized several residues in Atg8, including Arg²⁸, Tyr⁴⁹, and Leu⁵⁰, which are required for both interaction with Atg19 and regulation of general autophagic activity [192]. Additionally, residue Lys⁴⁸ and Leu⁵⁵ of Atg8 are also essential for autophagic transport, but are not required for Atg19 binding.

Sequence alignment of Atg8 and LC3 shows that these hydrophobic and positively charged residues are evolutionarily conserved, and are exposed to the surface. We thus hypothesize that the sorting mechanism of selective autophagy could also be evolutionarily conserved and mediated by sequences within LC3, which correspond to

essential residues in Atg8 for Atg8-Atg19 interaction. In the present study, we systematically examine the roles of conserved LC3 residues in its binding to p62 and its lipidation. The potential effect of LC3 mutants that lack p62 binding ability on the clearance of cytotoxic Huntingtin aggregates is also determined. Our findings provide the first direct evidence that the LC3-p62 interaction is pivotal for the selective recruitment of aggregation-prone proteins to autophagy, rendering neuroprotection against aberrant protein aggregates.

2.3 Materials and Methods Reagents

The QuikChange site-directed mutagenesis kit was from Stratagene. BCA protein assay reagent kit and ImmunoPure® Immobilized Protein A were purchased from Pierce. Mouse anti-p62 antibody was from BD Transduction Laboratories.

Mouse anti-GFP (B-2) antibody, rabbit anti-GAPDH (FL-335) antibody, rabbit anti-calnexin (H-70) antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, HRP-conjugated anti-rat IgG, and HRP-conjugated anti-mouse IgG were from Santa Cruz Biotechnology, Inc. Mouse anti-Htt antibody and Immobilon Western Chemiluminescent HRP Substrate were from Millipore Corporation. Earle’s balanced salt solution, saponin, poly-L-lysine, mouse anti-GFP antibody (clone GFP-20) (for

immunoprecipitation), and dimethyl pimelimidate dihydrochloride (DMP) were purchased from Sigma-Aldrich, Inc. Rapamycin was from CalBiochem.

Lipofectamine 2000 transfection reagent, DMEM, Alexa Fluor® 633 goat anti-mouse IgG (H+L), and 4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI) were from Invitrogen. Fetal bovine serum (FBS) was from Biological Industries Ltd (Kibbutz Beit Haemek, Israel). All other reagents were at least reagent grade and obtained from standard suppliers.

Site-directed mutagenesis of GFP-LC3 mutants

The pEGFP-C1-LC3 construct encoding wild-type LC3 was generated as previously described. Mutagenized pEGFP-C1-LC3 constructs encoding mutant LC3s (LC3-K30D, LC3-K51A, and LC3-K53A) were generated by QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. Primers

were designed by Primer-X website

(http://www.bioinformatics.org/primerx/index.htm). The sequences of paired primers for each mutant were as follows: K30D-forward (5'- GAG CAG CAC CCC ACC GAC ATC CCA GTG ATT ATA G-3') and K30D-reverse (5'-C TAT AAT CAC TGG GAT GTC GGT GGG GTG CTG CTC-3'); K51A-forward (5'-GTC CTG GAC AAG ACC GCC TTC CTT GTA CCT GAT C-3') and K51A-reverse (5'-G ATC AGG TAC

AAG GAA GGC GGT CTT GTC CAG GAC-3'); K53A-forward (5'-G GAC AAG ACC AAG TTC GCC GTA CCT GAT CAC GTG-3') and K53A-reverse (5'-CAC GTG ATC AGG TAC GGC GAA CTT GGT CTT GTC C-3'). The underlined nucleotides denote the base changes made to incorporate the desired missense mutations. The mutagenized sequences were confirmed by DNA sequencing.

To generate wild-type and mutant DsRed-tagged LC3 constructs, LC3 sequences were excised from pEGFP-C1-LC3 constructs by Eco RI and Bam HI. Purified DNA inserts were ligated with and subcloned into Eco RI/Bam HI-digested

pDsRed-Monomer-C1 vector (Clontech Laboratories, Inc. ).

Cell culture

Human embryonic kidney cells (HEK293) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. Cells were incubated in a humidified incubator at 37 ºC in 5% CO₂.

Transient transfection and induction of autophagy

Transient transfection of LC3 constructs into HEK293 cells was performed using Lipofectamine 2000 tansfection reagent as described by the manufacturers.

HEK293 cells were seeded onto 6-well microplates and transfected with 1 µg of

wildt-type or mutant pEGFP-LC3 constructs for 24 or 48 h to allow protein expression. To induce autophagy, following the removal of growth medium, transfected cells were washed by Earle’s buffered salt saline (EBSS) once and incubated in EBSS for 2 h. Cells incubated in fresh culture medium were included as controls. Alternatively, HEK293 cells transfected with different GFP-LC3s were treated with 0.2 µg/ml of rapamycin for various intervals as specified to induce autophagy. Clarified lysates derived from transfected cells were analyzed by SDS-PAGE and Western blotting.

To determine the autophagy-mediated clearance of Htt aggregates, HEK293 cells in 10-cm dish were transfected with 5 µg of pcDNA3.1-Htt-(Q)109-hrGFP (a generous gift from Dr. Yijuang Chern at Academia Sinica [193]) for 24 h.

Htt-(Q)109-hrGFP-transfected cells were subcultured onto 6-well microplates and

allowed to adhere overnight. Htt-(Q)109-hrGFP-expressing cells were then transfected with 0.5 µg/well of wild-type or mutant DsRed-LC3 constructs for 5h later.

Following the removal of transfection mixtures, cells co-transfected with Htt-(Q)109-hrGFP and DsRed-LC3 were incubated with culture medium in the presence of 0.1% DMSO or 0.2 µg/ml rapamycin for additional 48 h to induce autophagy.

The Generation of a tetracycline-inducible cell line that is stably transfected with YFP-tagged neomycin phosphotransferase II fusion construct

To generate an inducible neomycin phosphotransferase II-YFP (NeoR-YFP) construct, a marker protein for constitutive autophagy degradation [194], the coding sequence of NeoR was first subcloned into pEYFP-C1 (Clontech) in-frame with YFP tag. The NeoR-YFP cDNA sequence was subsequently subcloned into pcDNA5 plasmid (Invitrogen), and the expression of NeoR-YFP fusion protein would be under the control of tetracycline operator sequences. The NeoR-YFP construct was transfected into T-REx293 cells by lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. Transfected cells were cultured in DMEM supplemented with 10% FBS, 200 µg/ml hygromycin and 5 µg/ml blasticidin, and single colonies resistant to antibiotic selection were isolated individually. Each of independent cell lines was screened for the tetracycline-inducible expression of NeoR-YFP. Cell lines in which the accumulation of NeoR-YFP can be suppressed by

在文檔中 p62在蛋白質堆疊物的降解作用之調控機制 (頁 39-0)