REVIEW

Degradative proteomics and disease mechanisms

Francesco Lanucara1,2,3, Philip Brownridge2, Iain S. Young2, Phillip D. Whitfield2

and Mary K. Doherty1

1 The Physiological Laboratory, School of Biomedical Sciences, Faculty of Medicine, University of Liverpool, UK2 Proteomics and Functional Genomics Research Group, Faculty of Veterinary Science, University of Liverpool, UK3 Dipartimento di Chimica e Tecnologie del Farmaco, Universita di Roma La Sapienza, Italy

Received: July 29, 2009

Revised: September 22, 2009

Accepted: October 8, 2009

Protein degradation is a fundamental biological process, which is essential for the main-

tenance and regulation of normal cellular function. In humans and animals, proteins can be

degraded by a number of mechanisms: the ubiquitin-proteasome system, autophagy and

intracellular proteases. The advances in contemporary protein analysis means that proteo-

mics is increasingly being used to explore these key pathways and as a means of monitoring

protein degradation. The dysfunction of protein degradative pathways has been associated

with the development of a number of important diseases including cancer, muscle wasting

disorders and neurodegenerative diseases. This review will focus on the role of proteomics to

study cellular degradative processes and how these strategies are being applied to understand

the molecular basis of diseases arising from disturbances in protein degradation.

Keywords:

Autophagy / Degradative proteomics / Disease / Proteasome / Ubiquitin

1 Introduction

Maintaining cellular homeostasis in response to changing

environmental conditions requires a continual cycle of

synthesis and degradation of intracellular proteins. Protein

synthesis is closely related to the transcriptome, while

protein degradation is regulated by two main downstream

pathways, the ubiquitin-proteasome system (UPS) [1] and

macro-autophagy, typically referred to as autophagy [2, 3].

Short-lived proteins are believed to be degraded predomi-

nantly by the UPS and long-lived proteins and organelles are

degraded in the lysosome by autophagy [4]. However, it has

been observed that there may be overlap between autophagy

and proteasomal degradation and they may indeed be

compensatory [5]. Proteins can also be degraded by a range

of cysteine proteases [6, 7] that cleave proteins into smaller

fragments, which can then be further broken down by other

pathways.

Proteomic strategies are increasingly being used to probe the

structure and function of protein degradative pathways [8, 9].

Proteomics is a key technology for exploring these systems as it

has the ability to define changes in protein expression and

address the technically and conceptually challenging problems

of protein–protein interactions and PTMs. Investigators are also

beginning to adopt proteomic approaches to monitor the

dynamics of protein degradation in cells and complex organ-

isms [10–12]. In addition, proteomics is being used to under-

stand human diseases that result from the dysfunction of

protein degradative pathways. These include cancer [13, 14],

muscle wasting disorders [15, 16] and neurodegenerative

diseases [17, 18]. This review will focus on the role of proteo-

mics to study cellular degradation pathways and how these

strategies are being used to understand the molecular basis of

disease states arising from disturbances in protein degradation.

2 Protein degradative pathways

2.1 UPS

Proteasomal degradation is believed to be a protein-specific

pathway that permits the sensitive and rapid control of

Abbreviations: mdx, x-linked muscular dystrophy; MuRF1,

muscle-specific-RING-finger protein 1; SILAC, stable isotope

labelling of amino acids in cell culture; UPS, ubiquitin-protea-

some system

Correspondence: Dr. Mary K. Doherty, School of Biomedical

Sciences, University of Liverpool, Crown Street, Liverpool L69

3BX, UK

E-mail: doherty@liverpool.ac.uk

Fax: 144-151-794-4989

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com

Proteomics Clin. Appl. 2010, 4, 133–142 133DOI 10.1002/prca.200900159

cellular protein composition (Fig. 1). The 26S proteasome is

a multimeric complex (approximately 2.5 MDa), comprising

19S cap structures attached to either end of a barrel-shaped

20S core particle that contains the proteolytic machinery

[19]. In mammalian systems the 20S core particle is

composed of at least 14 subunits, designated a1–7 and b1–7.

The subunits are assembled into four stacked rings, each

containing seven subunits to form the cylindrical abba core.

While the a-subunits are required for structural arrange-

ment and regulation of substrate access, the b-subunits 1, 2

and 5, are catalytically active. The 19S cap is constituted

from at least 18 different subunits. These form a base that

sits adjacent to the 20S core and a lid, which is thought to be

involved in substrate recognition.

Ubiquitin is a small (8.5 kDa) protein and is the primary

regulator of protein degradation by the proteasome. Proteins

are targeted for proteasomal degradation by the attachment

of polyubiquitin chains [20]. A single ubiquitin molecule is

covalently attached via its C-terminal glycine residue to a

specific lysine residue on the substrate protein. This is

mediated by E2 ubiquitin-activating enzymes and multiple

E3 ubiquitin ligases. Ubiquitin chains are subsequently

formed primarily by linkage through Lys48. Recent work

has indicated that proteasomal degradation of proteins may

also be mediated through the linkage of polyubiquitin

chains at Lys11 and Lys63 [21]. Further, there are reports

that proteins are targeted for degradation by the UPS

through ubiquitin linkage to the N-terminal amino acid

[22–24] or by attachment to serine or threonine residues [25].

The polyubiquitinated proteins are then recognised by the

19S cap of the 26S proteasome, and are committed to

degradation in the internal hydrolytic cavity of the 20S

proteasome core. Once a protein has been committed to the

proteasome, degradation to the peptide and amino acid

components is rapid, with the amino acids free for future

protein synthesis.

2.2 Autophagy

Autophagy is involved in the removal of aged and damaged

proteins and organelles and is the major catabolic pathway

used to generate intracellular nutrients under periods of

stress. Autophagy is commonly stimulated in cells experi-

encing starvation and it is inhibited under conditions of

high nutrients. While autophagy is often described as being

the primary pathway for bulk cytosolic protein degradation,

it is increasingly being recognised as a more selective

process [26].

The degradation of proteins, macromolecules and cellu-

lar organelles by autophagy is a dynamic process in which

sub-cellular membranes undergo dramatic morphological

changes [27]. Portions of cytoplasm are sequestered within

double membrane vesicles described as autophagosomes

(Fig. 2). The autophagosome is then fused with the lyso-

some and the autophagic body is broken down. Although

the gross details of the autophagic process are known, the

signaling pathways that regulate and control autophagy

remain to be fully defined. One of the major pathways

involved in the induction of autophagic degradation is the

mammalian target of rapamycin pathway [28]. A number of

autophagy-related proteins from the mammalian target of

rapamycin pathway and their biological roles have been

characterised in mammalian cells and yeast [29] although

this is an area of ongoing investigation.

2.3 The cysteine proteases

Cysteine proteases are a family of degradative enzymes that

share a common mechanism of action. These enzymes

typically cleave proteins into smaller fragments that can

then be further degraded by other cellular degradative

pathways. The major cysteine proteases in mammalian

systems are the calpains and the caspases. Calpains

(calcium-dependent proteases with papain-like activity) are

expressed ubiquitously in humans and animals [30]. There

are two prototypical calpains designated m- and m-calpain,

which relates to the molar concentration of calcium required

for activation. Most calpains exert limited cleavage of

their substrates, which include the cytoskeletal proteins

myosin and spectrin. There does not appear to be a

conservation of sequence at the cleavage sites within the

identified substrates, with the majority of cleavages occur-

ring at unstructured areas of the proteins [31]. As well as

being regulated by intracellular calcium concentrations, the

Substrate

subunits subunits

19S Activator

20S Proteasome

26S Proteasome

Ub

E1

E1

E2 E2

SubstrateSubstrate

Ub

peptides

UbUb

UbUb

Ub

Ub

Ub

Ub

Ub

E3

Figure 1. Proteasomal degradation: One of the main pathways

for protein degradation is by the UPS. The 26S proteasome is

composed of a 20S core, capped by two 19S activator lids.

Proteins are targeted for degradation by ubiquitin. This is

attached to the substrate protein following a series of enzymatic

reactions performed by E1, E2 and E3 enzymes. Following the

attachment of a Lys48-linked polyubiquitin chain, the substrate

protein is recognised by the 19S cap, linearised and fed into the

20S core. The protein is proteolysed and the degradative

products released.

134 F. Lanucara et al. Proteomics Clin. Appl. 2010, 4, 133–142

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com

activity of the calpains is also modulated by the specific

inhibitor calpstatin.

Caspases (cysteine proteases that cleave proteins at a

cysteine-aspartic scissile bond) are synthesised as catalyti-

cally inactive proenzymes comprising a number of

domains [32]. Activation of each caspase is induced by

proteolytic cleavage between domains, resulting in the

assembly of the active enzyme complex. The caspases

generally proceed by a cascade mechanism and effect

their proteolytic action by binding the relevant substrate

via a conserved amino acid recognition site. Caspases

have been identified as proteolysing a range of proteins

from the cytoskeleton (e.g. actin and gelsolin), nucleus

(e.g. lamin A and B and associated receptors) and those

involved in cellular signalling such as protein kinases and

cytokines.

3 Understanding degradativemechanisms through proteomics

Proteomics provides a variety of powerful tools with which

to investigate protein degradative pathways. Considerable

research has focused on the UPS [33]; however, investigators

have also begun to apply proteomic techniques to the study

of autophagy [34] and the identification of the primary

cleavage substrates of calpains and caspases [35, 36].

Gel-based approaches have been shown to be of consid-

erable value in visualising degradative products of tissue

proteins [37], while MS strategies have been adopted to

probe the structure of the proteasome and develop models

of its assembly, activation and interaction [38–40]. MS has

also been employed to characterise ubiquitinated and

phosphorylated proteins and to determine the localisation of

these key PTMs. In many studies these analyses have

involved the isolation and enrichment of the proteins of

interest [41–43]. The modifications have then been identified

using bioinformatic searches, which incorporate the corre-

sponding mass shifts. Ubiquitinated peptides have been

putatively identified by searching for the added mass of a

GlyGly tag to a lysine residue [44]. This di-glycine tag is

derived from the tryptic cleavage of the ubiquitin–substrate

complex. However, caution should be taken when using this

approach as a similar mass shift has been observed as an

artefact of alkylation with iodoacetamide [45]. It is proposed

that the use of chloroacetamide in studies focussed on the

identification of ubiquitin modification would minimise

false positive rates as it shows greater selectivity for cysteine

residues. In the case of phosphorylated proteins, phosphate

groups can be easily fragmented in a mass spectrometer

using neutral loss or MS3 strategies [46]. A limitation of this

approach is that the position of the phospho-modification, in

general, has to be manually determined, particularly when

the peptide is multiply phosphorylated or when consecutive

residues are potential modification sites. This restricts the

use of these methods in global profiling experiments.

Recent developments such as electron transfer dissociation

and electron capture dissociation fragment the peptide to

give a different ion series compared with conventional

fragmentation methods [47]. In many instances this allows

labile modifications including phosphate groups to be

retained on the amino acid facilitating more robust assign-

ment of localisation.

MS analyses have also been employed for the quantifi-

cation of modified proteins. These include methods for

the chemical labelling of proteins, e.g. iTRAQ [48] and

stable isotope labelled internal standards, e.g. AQUA [49].

Experimental strategies using stable isotope labelling of

Starvation/extracellular stress Decrease in nutrients/block in nutrient uptake

Decreased intracellular nutrientsSignalling events

nucleation

pre-autophagosome autophagosome autophagolysosome

expansion fusion

lysosomedegradation

TCA cycle

amino acids protein synthesis

fatty acids

Figure 2. Autophagy: Protein degradation in

response to starvation and other extra-

cellular stress is predominantly by autop-

hagy. A decrease in nutrients causes

activation of signalling pathways. Autop-

hagy is initiated by the formation of double-

membraned vesicles that encapsulate the

cellular material to be degraded. This

autophagosome fuses with the lysosome to

form an autophagolysosome in which

breakdown occurs. Amino acids are then

recycled to allow further protein synthesis.

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amino acids in cell culture (SILAC)-based approaches have

been extended to permit multiplexed quantification and the

temporal analysis of PTM-modified protein populations [50].

More recently, SILAC has been adapted to allow the direct

quantification of protein synthesis and degradation rates

on a proteome-wide scale [51]. In this ‘‘dynamic SILAC’’

approach, the cultured cells are grown in the presence of

labelled amino acids until full incorporation is achieved. The

cells are then switched to media, which contains only light

amino acids and sampled over a period of time. The relative

loss of label over the ‘‘chase’’ period can be monitored by

MS allowing the rate of degradation to be calculated for

hundreds of proteins. The ability to accurately define rate

constants of degradation on a global scale presents an

exciting prospect for future research into diseases that are

influenced by degradative processes.

4 Protein degradation and disease

Damaged proteins can form aggregates and the prompt

removal of these proteins is essential in maintaining normal

cellular function. Disturbances of protein degradative

systems can therefore lead to the development of a variety of

disease states [52, 53]. The following section highlights

some key examples where proteomic approaches have been

used to investigate diseases that are linked to altered protein

degradation. The research presented aims to demonstrate

how proteomic technologies are providing a more compre-

hensive view of the cellular control mechanisms involved in

these complex pathologies.

4.1 Cancer

Cancer encompasses a group of diseases, which are char-

acterised by uncontrolled growth of cells, invasion of

adjacent tissues, and in many cases the dispersal of

the cells to other locations of the body, metastasis [54].

Proteomics has been used extensively to characterise

the relative differences in protein expression between

cancerous and non-cancerous tissues samples. In one such

investigation, a gel-based approach compared the protein

expression between normal and breast cancer tissues. The

study showed that the abundance of ubiquitin-specific

proteases, proteasome subunits and E3 enzymes was

increased in the cancerous tissue. This indicated that there

was a marked up-regulation in the activity of the UPS in the

disease state [55].

Other cancer proteomic studies have focussed on the role

of the UPS in metastasis. Zhang et al. [56] analysed three

mouse lung adenocarcinoma cell lines with different

metastatic potential. The cells were grown in the presence of35S-methionine and collected at three different time-points.

Proteins were then separated by 2-DE and quantified using

autoradiography. A comparison of the cellular proteomes

revealed that a number of proteins were differentially

expressed, which were then identified by MALDI-MS/MS.

Among these, 28 proteins showed a systematic trend

corresponding to metastatic potential. Bioinformatics

analysis indicated that several of these proteins were

involved in proteasome, cell-cycle and cell communication

pathways. These included keratins, 14-3-3 proteins and

components of the 26S proteasome whose aberrant expres-

sion may be directly or indirectly involved in cancer devel-

opment and metastasis.

A disturbance in autophagy has also been proposed to

play a role in the pathogenesis of cancer. An up-regulation

of autophagy in tumour cells could maintain the viability

and therefore survival of the cancerous cells. Indeed,

autophagy has been reported to be markedly increased

during the tumour formation [57]. Interestingly, tumour

cells can display a deficiency in autophagic degradation

[58, 59]. The impairment of autophagy may also affect the

response of cells to chemotherapy or radiotherapy by redu-

cing the ability of the cell to remove proteins or organelles,

which may have been damaged by the treatment [60, 61]. A

consensus role for autophagy in cancer development is still

lacking, [62] and in this regard proteomic studies may be

helpful in defining the specific correlation between autop-

hagic aberrations and distinct types of cancer.

In a recent study, DIGE was used to probe the link

between defective autophagy and the promotion of

tumourigenesis in immortalised baby mouse kidney cells

[63]. The authors observed that in autophagy-defective cells

subjected to metabolic stress there was an accumulation of

p62, an adaptor protein involved in the targeting of poly-

ubiquitinated proteins to the autophagosome membrane.

There is evidence to suggest that increased expression p62 is

linked to the promotion of tumourigenesis [64]. Moreover,

p62-deficient cells show reduced carcinogenic potential [65].

The study also showed that ER chaperones and proteins

from the protein disulfide isomerase family were elevated.

These proteins are involved in protein refolding, indicating

that in tumour cells there is a deficiency in the management

of protein turnover.

4.2 Diseases related to muscle wasting

Many disease states such as starvation, diabetes mellitus and

sepsis are characterised by accelerated proteolysis of skeletal

muscle. The loss of muscle mass is inextricably linked to

loss of protein, in particular myofibrillar proteins. In muscle

wasting, the increase in proteolysis occurs primarily via the

UPS although the other degradative pathways are involved,

especially where the muscle wastage is rapid [66].

Muscle-specific-RING-finger protein 1 (MuRF1) is an

ubiquitin ligase that is up-regulated during various muscle

wasting conditions. Although the substrates of this enzyme

are not fully defined, MuRF1 binds to large myofibrillar

proteins [67]. To test the hypothesis that MuRF1 promotes

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muscle atrophy, transgenic mouse models over-expressing the

protein were generated [68]. A DIGE comparison of quad-

riceps from the transgenic and wild-type mice failed to reveal

an increase in the multi-ubiquitination of myosin, which has

previously been observed in human patients with muscle

wasting. However, pyruvate dehydrogenase, and its regulator,

PDK2 as well as phosphorylase b and glycogenin were

suppressed. These findings suggest that MuRF1 expression in

skeletal muscle is intimately involved with skeletal muscle

carbohydrate metabolism during metabolic stress.

Muscle wasting is also a feature of neuromuscular

disorders such as Duchenne muscular dystrophy. The lack

of dystrophin is known to initiate the loss of a range of

surface proteins including dystroglycans and sarcoglycans

in this disease state; however, the secondary effects on

global protein expression are poorly defined. To explore

these changes, Ge et al. [69, 70] analysed the proteome of

hind leg muscle from x-linked muscular dystrophy (mdx)

mice at different stages of the disease (1, 3 and 6 months) in

two related studies. Muscle proteins were separated by 2-DE

and identified using MALDI-QTOF. Comparison of the mdx

and control muscle revealed marked differences between the

overall protein patterns. Sixty proteins were found to be

differentially expressed, of which 40 were cytosolic and 20

were microsomal proteins. The authors noted that in the

3- and 6-month old mice, the majority of these proteins were

up-regulated, while at 1 month the same proteins were

down-regulated, which reflected changes in the disease

progression. Of particular note, there was a significant

decrease in the expression of adenylate kinase in mdx mice

(more than fourfold down-regulated). Adenylate kinase is an

important protein in the synthesis and regulation of

nucleotides and this protein may play a critical role in the

impaired regulation of energy metabolism in Duchenne

muscular dystrophy. Myosin light chain 2 was consistently

up-regulated in the mice at all ages and it was suggested that

this may be due to an abnormal state of differentiation in

the disease state.

Another proteomics study compared the diaphragm

muscle from control and mdx mice [71]. A total of 2398

individual protein spots were visualised with 35 being

differentially expressed. Of the proteins that were up-regu-

lated, cardiovascular heat shock protein was shown to have

an eightfold increase in concentration. This finding was

confirmed by Western blotting and was found to be more

pronounced as the mice aged. In addition, visualisation by

immunofluorescent microscopy indicated that the protein

was associated with cytoskeletal components of the muscle

fibre. It was postulated that the protein is involved in the

stress response to damaged fibres and may play an impor-

tant protective role. Interestingly, the Fbxo11 protein, the

substrate recognition component of an E3 ubiquitin–protein

ligase complex involved in ubiquitination, was found to be

down-regulated. A decrease of this protein supports the

hypothesis that the UPS is impaired in muscle-wasting

diseases.

Muscle wasting is also observed in sepsis; however, to

date, few proteomic studies have examined the biochemical

changes associated with this condition. One study has

employed a rat model of cutaneous burn injury with

superimposed infection to investigate changes in protein

expression in skeletal muscle [72]. The only protein that

showed a substantial up-regulation was myosin binding

protein-H. This protein has a strong affinity for myosin with

putative roles in the assembly of myofibrils. As the

concentration of myosin was greatly reduced, it was

proposed that myosin binding protein-H may play a role in

myosin degradation. A number of chaperone proteins,

e.g. HSPb6 and metabolic enzymes including ATP synthase

b-chain were found to be down-regulated, which the authors

suggested could lead to a concomitant decrease in global

protein synthesis following severe injury.

Aging is often accompanied by muscle wasting, a

condition known as sarcopenia. There can be a loss in

muscle mass, arising from both a decrease in the number of

muscle fibres and the atrophy of individual fibres. Structural

and chemical changes in myosin during contraction have

been linked to age-related decreases in specific force and

inhibition of contractility [73]. Aged muscle may also be

more susceptible to oxidative damage, which may contribute

to impaired muscle performance. DIGE has been used to

examine the differences in proteome between skeletal

muscle from young (20–30 years) and elderly, active

(75 years) subjects [74]. A number of proteins involved in

oxidative metabolism were found to be more abundant in

elderly muscle whereas enzymes catalysing reactions

involved in anaerobic metabolism, e.g. creatine kinase,

glycolytic enzymes and transport proteins were of lower

concentration. The expression profile of contractile proteins

was also altered. In elderly individuals there was a decrease

in the expression of the phosphorylated ‘‘fast’’ forms of

myosin light chain kinase. The authors commented that

these changes in expression may be linked to impaired

performance and increased fatigue.

4.3 Neurodegenerative diseases

Deregulation of protein degradative pathways has been

implicated in the pathogenesis of many neurodegenerative

disorders [75, 76]. A key feature of the pathology of many of

these diseases is the formation of insoluble protein aggre-

gates in the brain. The plaques can be made up from

mutant, misfolded or damaged intracellular proteins, which

degradative processing has failed to remove. The case for

examining autophagy in neurological disease states has

been driven by the observation that the disruption of crucial

autophagy genes in mice leads to the accumulation of

polyubiquitinated proteins, which may be particularly

harmful for neurons [77, 78]. Autophagic vacuoles contain-

ing disease-related proteins have also been observed in

regions of the brain affected by Alzheimer’s disease [79].

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The UPS is also reported to play a critical role in neurolo-

gical disorders. A mouse model in which a component of

the 26S proteasome, PSMC1, was knocked out showed

impairment of protein degradation by the UPS and forma-

tion of neuronal Lewy-like inclusions [80].

Axonal degeneration is a common pathological feature

of many degenerative disorders; however, the underlying

molecular mechanisms have not yet been fully elucidated.

In a recent study, Goto et al. [81] employed a proteomics

approach to examine protein expression in sciatic nerves

in gracile axonal dystrophy mice. This mouse model used

is a knock-out of ubiquitin C-terminal hydrolase L1, a

de-ubiquitinating enzyme, which has been shown to stabi-

lise mono-ubiquitin in neurons. Depletion of ubiquitin

C-terminal hydrolase L1 leads to a decrease in mono-

ubiquitin and an impairment of proteasomal degradation.

Using DIGE, there was an age-dependent accumulation of

several proteins including GAPDH, 14-3-3, annexin V and

neurofilament L observed in gracile axonal dystrophy mice

compared with wild-type animals. GAPDH, and the

sulphonated form of the protein, was found to aggregate in

axons of the mouse model, indicating an oxidative stress

response.

Proteomic studies have been used to probe the link

between the UPS and Parkinson’s disease. S-nitrosylation of

cysteine residues in the E3 ubiquitin ligase parkin has been

observed both in animal models and in human brain

samples [82]. The PTM leads to an initial promotion of the

E3 ligase activity, resulting in autoubiquitination of parkin

itself. This autoubiquitination in turn results in down-

stream inactivation of the ligase and subsequent proteasome

dysfunction, aberrant protein accumulation and ultimately,

cell death. Moreover, parkin has been shown to be mutated

in Parkinson’s disease [83].

The relevance of PTMs of the 20S components has also

been investigated in Alzheimer’s and normal brain tissues.

Gillardon et al. [84], using DIGE and tandem MS, found that

the a7, a4, b2 and b7 subunits differed in their relative

abundances and showed changes in phosphorylation and

acetylation status. Although the functional relevance of

these modifications is not fully understood, it is possible

that they are involved in the interaction of the 20S subunits

with other proteins. In the case of Alzheimer’s, modification

of the a4 subunit may alter the interaction of the protea-

some with the amyloid peptide and contribute to the aber-

rancies of the neuronal amyloid-b processing [85].

The role of phosphorylation and polyubiquitination in

the impairment of the degradative pathways in neurological

diseases was the focus of another proteomic study on the

microtubule-associated protein Tau [86]. Tau is part of the

degradation-resistant core of neurofibrillary tangles along

with the senile plaques of amyloid-b peptide. The protein

was isolated from brain specimens from patients with

Alzheimer’s and LC-MS/MS was used to analyse the affinity

purified Tau. The study showed that the protein was (poly)-

ubiquitinated at six different identified lysine residues.

These binding sites were localised to the microtubule

binding domain. The polyubiquitin chains were found to be

either Lys48 or Lys6, both of which are involved in protein

degradation. Lys48 polyubiquitination is well-recognised as

the signal of targeting proteins for degradation by the UPS.

It has also been reported that modifications of Lys6 inhibits

ubiquitin-dependent protein degradation [87]. Therefore, the

identification of these modifications indicates that dysfunc-

tion of the UPS may initiate the formation of degradation-

resistant polyubiquitinated Tau tangles in Alzheimer’s. The

finding that Tau is ubiquitinated but not degraded in

Alzheimer’s disease has been confirmed in a proteomic

study by Riederer et al. [88] in which post-mortem brain

samples from middle-aged and elderly subjects with and

without Alzheimer’s disease were analysed.

A recent study has used an MS-based strategy to quantify

polyubiquitin chains in Huntington’s disease [89]. It was

shown that Lys48-linked polyubiquitin chains accumulated

in the brains from both a mouse model and human patients

with Huntington’s disease. Moreover, the accumulation of

other polyubiquitin chains, namely Lys63 and Lys11-linked,

indicated that more global changes in the ubiquitin system

are involved.

4.4 Lysosomal storage disorders

Lysosomes are important degradative compartments of

eukaryotic cells. Defects in lysosomal function are associated

with a group of over 50 inherited diseases known as the

lysosomal storage disorders. Clinically, these diseases

present with a diverse range of symptoms such as progres-

sive neurodegeneration, organomegaly and skeletal

abnormalities. The majority of lysosomal storage disorders

are caused by the deficiency of a single acid hydrolase;

although they can also result from the dysfunction of the

cellular machinery involved in lysosomal protein trafficking

and defective transporters that move the products of lyso-

somal catabolism across the lysosomal membrane.

There has been considerable focus in the application of

proteomic technologies to characterise the soluble and

membrane proteomes of the lysosome [90–92]. Proteomic

approaches have also been used to determine disease-

related disturbances in the protein profiles of body fluids

and tissues from patients with lysosomal storage disorders.

In a pilot study, a label-free LC-MS strategy was adopted to

define changes in the serum protein expression in patients

with type I Gaucher disease [93]. The analyses revealed

alterations in the concentrations of fibrinogens, comple-

ment cascade proteins and high-density lipoproteins in

affected individuals. The differential labelling of proteins

has been used to compare changes in the serum of

Fabry patients before and after a 6-month period of

enzyme replacement therapy [94]. Statistically significant

decreases in the relative concentrations of a2-HS glycopro-

tein, vitamin D-binding protein, transferrin, Ig-a-2 C chain

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and a-2-antiplasmin were observed following enzyme

replacement therapy. More recently, Sleat et al. [95] analysed

mannose-6-phosphorylated proteins, which had been puri-

fied from brain autopsy samples of patients with suspected

lysosomal storage disorders. The authors reported that

spectral counting of peptides detected by MS/MS was able to

provide definitive diagnosis in 8 out of 23 cases and

suggested that this approach may prove to be a comple-

mentary tool to conventional methods used in charactering

lysosomal diseases.

Although a great deal of progress has been made in the

understanding of the metabolic and molecular basis of

lysosomal storage disorders, the mechanisms by which

lysosomal storage leads to cellular dysfunction have yet to be

fully elucidated. There has been an increasing interest in the

possible involvement of autophagy in the pathogenesis of

lysosomal storage disorders [96]. Studies have revealed that

in specific lysosomal diseases there is a deficiency in the

fusion of autophagosomes and lysosomes, leading to the

accumulation of protein aggregates and impaired organelle

turnover. These include Pompe disease [97], mucolipidosis

type IV [98], mucopolysaccharidosis type IIIA and multiple

sulfatase deficiency [99]. The investigation of defective

autophagy in lysosomal storage disorders is still emerging

and proteomic strategies offer the opportunity to further

explore this link and provide additional insights into the

pathophysiology of these disease states.

5 Concluding remarks

Cells continuously adapt to changing environmental condi-

tions by adjusting their protein content to prevailing needs.

This homeostasis involves continuous biosynthesis and

degradative processes. Efficient protein degradation is

essential for the maintenance of cellular health as damaged

proteins commonly develop abnormal intermolecular

interactions resulting in the formation of aggregates. As

such the prompt degradation of irreversibly damaged

proteins is essential to preserve normal cellular function. A

number of disease states have been linked to the malfunc-

tion of cellular degradative pathways, in particular the UPS

and autophagy. While much is known about the regulation

of these pathways under normal conditions in cell culture

system, it is only recently that we are beginning to combine

clinical understanding with fundamental biochemical

knowledge. In many instances, for example, it is not clear

whether the degradative malfunction is a direct cause or a

downstream effect of the disease.

Proteomics is a key strategy in defining the role of

degradative pathways in disease processes. With more

recent advances in proteome quantification and character-

isation of PTMs, it is becoming possible to accurately define

changes in protein pathways due to disease states. In

combination with methods to accurately define individual

protein degradation rates on a large scale, we are moving to

a robust, global understanding of how the cells degradative

mechanisms interact and function in the normal cell and in

diseased conditions. In future, it will be important to inte-

grate data from proteomic-based investigations with other

‘‘omic’’ technologies and with clinical data to fully under-

stand the underlying pathology and biochemistry of these

varied diseases and to develop targeted therapies.

The authors wish to acknowledge the RCUK, The RoyalSociety and BBSRC for supporting research in their laboratories.

The authors have declared no conflict of interest.

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