DOI: 10.2478/s11535-006-0017-3Rapid communicationCEJB 1(2) 2006 289–298

Proteasome activity in experimental diabetes

Albena Alexandrova∗, Lubomir Petrov, Margarita Kirkova

Institute of Physiology,Bulgarian Academy of Sciences,

1113 Sofia, Bulgaria

Received 13 March 2006; accepted 24 April 2006

Abstract: Numerous studies have indicated that oxidative stress contributes to the developmentand progression of diabetes and other related complications. Since the ubiquitin-proteasome pathwayis involved in degradation of oxidized proteins, it is to be expected that alterations in proteasome-dependent proteolysis accompany diabetes. This paper focuses on the role of the proteasome in alloxan-induced experimental diabetes. The changes in proteasomal activity and oxidative stress indices (proteinoxidation and lipid peroxidation) were evaluated. The obtained results revealed increased proteinoxidation and lipid peroxidation, as well as alterations in proteasomal activities in diabetic rats. Ourdata indicates a significant decrease in chymotryptic-like activity; increased tryptic-like activity; andunchanged post-glutamyl peptide hydrolytic-like activity. These findings suggest the presence of oxidativestress in diabetes that appears to result in changes to the ubiquitin-proteasome pathway.c© Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.

Keywords: Proteasomes, proteasome activities, diabetes, protein oxidation, rats

1 Introduction

Proteasomes are non-lysosomal, multicatalytic proteinase complexes present in a range

of organisms from bacteria to mammals [1]. They are located in both the cytoplasm

and nucleoplasm [2] and account for up to 1 % of total cellular protein. A proteasome

is a cylindrical 20S complex [3, 4], and in contrast to lysosomes, proteasomes deal pri-

marily with endogenous proteins such as transcription factors, cyclins, proteins that are

encoded by failure genes, incorrect or misfolded proteins or those damaged by other cy-

tosolic molecules. Although the 20S proteasome is described as a complex with multiple

peptidase activities, five specific activities have been defined [5, 6]: a chymotryptic-like

∗ E-mail: a alexandrova bas@yahoo.com

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(ChT-L) activity, a tryptic-like (T-L) activity, a post-glutamyl peptide hydrolytic-like

(PGPH) activity, an activity which cleaves the peptide bond after branched-chain amino

acids (BrAAP activity), an activity, which cleaves the peptide bond after small neutral

amino acids (SNAAP activity). The best-characterized of these peptidase activities are

the ChT-L, T-L and PGPH.

The 20S proteasome catalyzes the hydrolysis of peptides and larger proteins in an

ATP-independent fashion. This form of proteasome can associate with two regulatory

complexes, forming the 26S proteasome, which acts in concert with ubiquitin to form the

ubiquitin–proteasome system (UPS). As a principal mechanism for protein catabolism

in the cytosol and nucleus of mammals, UPS is involved in almost all cellular processes.

These include proliferation, differentiation, cell cycling, apoptosis [7], immune response

and inflammation [8]. Therefore, it is not unexpected that a significant number of human

diseases has been directly or indirectly associated with abnormalities in the UPS.

Diabetes mellitus is a chronic, progressive disease determined by environmental and

genetic factors. Numerous studies indicate that oxidative stress contributes to the de-

velopment and progression of diabetes and other related complications. Since UPS is

involved in the degradation of oxidized proteins it may be expected that alterations in

proteasome-dependent proteolysis can accompany diabetes. In diabetes there are multi-

ple sources of oxidative stress, including both enzymatic and non-enzymatic pathways.

For examples hyperglycemia may cause a huge production of free radicals, generated in

direct glucose auto-oxidation [9]. In addition non-enzymatic and progressive glycation

of proteins leads to the development of Amadori products, followed by formation of ad-

vanced glycation end-products, both of which generate ROS are at multiple stages [10].

The enzymatic sources of oxidant species, and subsequent oxidative stress, include the

activation of NAD(P)H oxidases [11], nitric oxide synthase [12], and xanthine oxidase

[13].

Toxic, reactive oxygen-derived free radicals play a crucial role in diabetes [14]. They

are able to attack all cellular macromolecules – including lipids, proteins, DNA, carbo-

hydrates. The oxidative attack of proteins can covalently modify amino acid residues,

leading to a variety of products [15]. Introduction of carbonyl groups, such as aldehydes

and ketones, into amino acid structures is a frequent consequence of protein oxidation.

Therefore, carbonyl groups are commonly accepted as an oxidative modification marker

[15]. In vitro data demonstrates that lipid peroxidation, but not glycation, leads to car-

bonyl modification in proteins [16]. Since lipid peroxidation is increased in diabetes, this

should result in an increase in protein carbonyl oxidative damage. On the other hand, it

has been shown that mild oxidation of proteins increases their susceptibility to proteolysis

by proteasomes. In contrast, severe oxidative stress diminishes intracellular proteolysis,

probably by generating heavily injured cell proteins (e.g. cross-linked/aggregated pro-

teins) that cannot be easily degraded, and also by damaging proteasomes [17, 18].

Thus the aim of the presented study was to evaluate changes in lipid peroxidation,

protein carbonyl contents and proteasomal activity (in particular T-L, ChT-L and PGPH

activity) in rats with alloxan-induced diabetes.

A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298 291

2 Materials and methods

2.1 Materials

Alloxan was purchased from Fluka Chemie GmbH (Germany). Suc-Leu-Leu-Val-Tyr-

7-amido-4-methylcoumarin (LLVY-AMC), N-t-Boc-Leu-Ser-Thr-Arg-7- amido-4-methyl-

coumarin (LSTR-AMC), N-Cbz-Leu-Leu-Glu-β-naphthylamide (LLE-NA), 7-amino-4-me-

thylcoumarin, β-naphthylamide and the proteasome inhibitor MG-132 (N-Cbz-Leu-Leu-

Leucinal) were all purchased from Sigma-Aldrich Chemie GmbH (Germany).

2.2 Animals

Male Wistar rats, weighing 180-200 g, were randomly divided in two groups: control and

alloxan-treated (n=6, for each group). The animals were housed in an air-conditioned

room at 22 ◦C and fed standard rat chow and water ad libitum. In the experimental group,

each rat was injected with a single dose of alloxan (120 mg/kg bw intraperitoneal), while

the control animals received ip saline. After 7 days the rats were fasted overnight and

then used in the experiments described herein. All measurements were done in triplicate,

the total number of the animals used being 36.

All experiments were performed in accordance with the “Principles of laboratory an-

imal care” (NIH publication No. 85-23, revised 1985), and the rules of the Ethics Com-

mittee of the Institute of Physiology, Bulgarian Academy of Sciences (registration FWA

00003059 by the US Department of Health and Human Services).

3 Experimental procedures

In rats under light ether anaesthesia, the left heart ventricle was punctured, blood was

taken with a heparinized syringe and centrifuged for 10 min at 3000 rpm to obtain blood

plasma.

Plasma glucose content was measured by the Glucose-PAP enzymatic-colorimetric

method (ABX Diagnostics, France) and was expressed in mg/dl. Only rats with more

than 200 mg/dl glucose were further used in the study.

Liver perfused with cooled 0.15M KCl was used to obtain a 10 %-homogenate in 0.15M

KCl. This was centrifuged for 10 min at 600 g to obtain a “post-nuclear” homogenate. A

further portion was centrifuged at 100 000 g to obtain a cytosolic fraction.

3.1 Analytical methods

Protein content was measured by the method of Lowry et al. [19].

Lipid peroxidation (LPO) was determined by the amount of thio-barbituric acid re-

active substances (TBARS) formed in fresh liver homogenates after a 60-min incubation

at 37 ◦ [20]. The A600 was considered to be a nonspecific baseline and was subtracted

292 A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298

from A532. The values were expressed in nmoles malondialdehyde (MDA) per mg protein,

using a molar extinction coefficient of 1.56 × 105M−1cm−1.

Protein carbonyl was quantified by reaction with 2,4-dinitrophenylhydrazine

(2,4-DNPH), according to Whitekus et al. [21], with some modifications. 0.5-ml samples

(1 mg/ml) were incubated with 1.5 ml 10 mM 2.4-DNPH (in 2M HCl) at room tempera-

ture for 1 h. After addition of 1 ml 40 % TCA the samples were centrifuged for 3 min at

11 000 g. The pellets were washed 3-times with ethanol-ethyl (1:1 v/v) and dissolved in

6M guanidine (in 20 mM potassium phosphate buffer, pH 2.3, adjusted with HCl). The

absorbance at 366 nm was measured and the carbonyl content was expressed in nmoles

carbonyl/mg protein, using a molar extinction coefficient of 2.2 × 104M−1cm−1.

Peptidase activity of proteasomes was measured fluorimetrically according to Bul-

teau et al. [22] using a Perkin-Elmer MPF-44B fluorimeter. Assays were performed in

the presence (20 μM) and absence of the proteasome inhibitor MG-132 (N-Cbz-Leu-Leu-

Leucinal). Any difference established was attributed to proteasome activity. Cytosolic

liver fractions were assayed for T-like activity, ChT-like activity and PGPH activity using

the fluorogenic peptides LSTR-AMC, LLVY-AMC and LLE-NA respectively. Fluores-

cence of the leaving group 4-amino-methyl-coumarin for LLVY-AMC and LSTR-AMC

was measured at 365 nm (excitation) and 465 nm (emission). Fluorescence of the leaving

group β-Naphtylamine for LLE-NA was measured at 335 nm (excitation) and 410 nm

(emission). Values were expressed in fluorescent units (FU) per mg protein.

4 Statistic analysis

The results were statistically analyzed by one-way ANOVA (with Dunnett post-test) with

p < 0.05 being accepted as the minimum level of statistical significance.

5 Results

7 days after alloxan treatment, the plasma glucose in the experimental group was in-

creased 3-fold, in comparison with the control group, 255±19.2 mg/dl vs. 82.0±2.7

mg/dl respectively.

In liver homogenates of rats with alloxan-induced diabetes the markers of lipid per-

oxidation (TBARS) and protein oxidation (carbonyl content) were increased as shown in

Fig. 1.

Analysis of the degradation of proteasome substrates LLVY-AMC, LSTR-AMC and

LLE-NA, respectively, indicates a significantly decreased ChT-L activity, increased T-L

activity and unchanged PGPH activity (Fig. 2).

A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298 293

Fig. 1 The content of TBARS (A) and carbonyls (B) in liver homogenate from diabetic

rats. The values represent the mean ±SEM of six animals per group. ∗P<0.05.

6 Discussion

It has been proposed that proteasomes play an important biological role [23], such as

forming a secondary antioxidant defense system or destroying oxidised proteins that are

potentially cytotoxic [24]. It has been shown that the extent of oxidative stress has a

varying effect on proteasome activity [25].

Oxidative stress and oxidative tissue-damage are the common end-point of a variety

of diseases, including diabetes. Results from a variety of studies have shown that diabetes

mellitus may alter proteasome activity, however this data is often controversial. Merforth

et al. [26] found a reduction in overall proteasome activity in the muscle extracts of rats

with streptozotocin-diabetes. However according to other authors, diabetic rats have

higher rates of proteolysis when compared with non-diabetic rats [27, 28] as a result of

hypoinsulinemia-induced activation of UPS. In contrast to Runnels et al. [29], Hayasi

and Faustman [30] revealed the presence of a specific proteasome defect in non-obese,

diabetic mice.

294 A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298

Fig. 2 Peptidase specific activities of liver proteasomes from control and diabetic rats:

(A) Chymotryptic-like, (B) Tryptic-like, and (C) PGPH. Activities are expressed as flu-

orescent units per milligram of protein. The values represent the mean ±SEM of six

animals per group. ∗P<0.05.

In the present study the relationship between diabetes-induced oxidative stress and

proteasomal activity was examined in alloxan-treated rats. We found an increase of TBRS

and protein carbonyls in the rat liver. Our data suports the finding of Rhee et al. [31] who

have found increased TBARS and carbonyl content in Streptozotocin-induced diabetic

rats. Jang et al. [32] also detected significantly increased levels of MDA and carbonyls

in liver, kidney and pancreatic mitochondria in streptozotocin-treated rats.

A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298 295

In other tissue preparations, alterations in protein and lipid status have also been de-

tected. Altomare et al. [33] noted an increased formation of protein-bound free sulfydryls

and carbonyl proteins in the eyes of diabetic patients. Significant increases in both lipid

peroxidation and protein carbonyl content confirm the occurrence of oxidative damage in

the intestinal mucosa of diabetic rats [34].

It is known that increased LPO can lead to a rise in protein oxidative damage, and

the production of toxic and reactive aldehyde metabolites; with MDA and 4-hydroxy-

2-nonenal (HNE) being the most important. It has been shown that these metabolites

are able to form covalent links with various molecules, including proteins [35]. The

conversion of some amino acid residues to carbonyl derivatives has also been observed

[36]. Mammalian cells have limited direct repair mechanisms, and most oxidized proteins

undergo selective proteolysis, a process that is frequently mediated by the proteasome.

In most cells oxidized proteins are degraded by the 20S proteasome in an ATP- and

ubiquitin-independent pathway [24]. However, some studies indicate the involvement of

both ATP/ubiquitin-independent and ATP/ubiquitin-stimulated pathways [37].

Since it has been shown that the mild oxidation of proteins increases their susceptibil-

ity to proteolysis by proteasomes [17], we measured T-L, ChT-L and PGPH proteasomal

activity in alloxan-induced diabetes. Our results indicate a significantly decreased ChT-L

activity, increased T-L activity and unchanged PGPH activity. These findings suggested

increased protein oxidative damage and alterations in the cytosolic proteolytic pathways

in this experimental model of diabetes. Analysis of proteasome substrate (LLVY-MCA)

degradation, Portero-Otin et al. [38] also reported a significantly decreased chymotryptyc

activity in both the liver and kidney of diabetic rats. The loss of proteasome activities

could be partially ascribed to lipid peroxidation end-products [39], suggesting impairment

of ubiquitin-proteasome proteolysis by accumulation of oxidatively modified proteins.

In addition, UPS is involed not only in the destruction of oxidatively-modified pro-

teins, but also in the degradation of regulatory proteins, enzymes and transcription

factors. A recent study involving the inappropriate degradation of insulin signalling

molecules in experimental diabetes suggested that altered UPS might be one of the molec-

ular mechanisms of insulin resistance [40]. Kawaguchi et al. [41] have shown that UPS

plays an essential role in the normal regulation of insulin secretion.

The present study revealed increased TBARS and protein carbonyls in the liver of

alloxan-diabetic rat. Diverse changes in ChT-L, T-L and PGPH proteasome activities

were also found. The obtained results could give no definite answer as to whether the es-

tablished changes in proteolytic activity were a direct consequence of developing oxidative

stress or a result of other diabetes-related metabolic alterations. Assigning a definite role

for proteaseme activities in the genesis and progression of diabetes and its complications,

is worthy of future investigations.

Acknowledgment

This work was supported by Grant L-1522 of the National Science Fund, Sofia, Bulgaria.

296 A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298

References

[1] P. Brooks, G. Fuertes, R.Z. Murray, S. Bose, E. Knecht, M.C. Rechsteiner, K.B.

Hendil, K. Tanaka, J. Dyson and J. Rivett: “Subcellular localization of proteasomes

and their regulatory complexes in mammalian cells”, Biochem. J., Vol. 346, (2000),

pp. 155–161.

[2] C. Wojcik and G.N. DeMartino: “Intracellular localization of proteasomes”, Int. J.

Biochem. Cell Biol., Vol. 35, (2003), pp. 579–589.

[3] J. Lowe, D. Stock, B. Jap, P. Zwickl, W. Baumeister and R. Huber: “Crystal struc-

ture of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution”,

Science, Vol. 268, (1995), pp. 533–539.

[4] G. Puhler, S. Weinkauf, L. Bachmann, S. Muller, A. Engel, R. Hegerl and W.

Baumeister: “Subunit stoichiometry and three-dimensional arrangement in protea-

somes from Thermoplasma acidophilum”, EMBO J., Vol. 11, (1992), pp. 1607–1616.

[5] M. Orlowski, C. Cardozo and C. Michaud: “Evidence for the presence of five distinct

proteolytic components in the pituitary multicatalytic proteinase complex. Proper-

ties of two components cleaving bonds on the carboxyl side of branched chain and

small neutral amino acids”, Biochem., Vol. 32, (1993), pp. 1563–1572.

[6] H.P. Schmid and J. Briand: “Proteasomes and related complexes”, Mol. Biol. Re-

ports, Vol. 24, (1997), pp. 1–138.

[7] C. Naujokat and S. Hoffmann: “Role and function of the 26S proteasome in prolif-

eration and apoptosis”, Lab Invest., Vol. 82, (2002), pp. 965–980.

[8] E. Reinstein: “Immunologic aspects of protein degradation by the ubiquitin-

proteasome system”, Isr. Med. Assoc. J., Vol. 6, (2004), pp. 420–424.

[9] S.P. Wolff and R.T. Dean: “Glucose autoxidation and protein modification. The

potential role of ’autoxidative glycosylation’ in diabetes”, Biochem. J., Vol. 245,

(1987), pp. 243–250.

[10] M. Brownlee, A. Cerami and H. Vlassara: “Advanced products of nonenzymatic

glycosylation and the pathogenesis of diabetic vascular disease”, Diabetes Metab.

Rev., Vol. 4, (1988), pp. 437–451.

[11] T. Inoguchi, P. Li, F. Umeda, H.Y. Yu, M. Kakimoto, M. Imamura, T. Aoki, T.

Etoh, T. Hashimoto, M. Naruse, H. Sano, H. Utsumi and H. Nawata: “High glucose

level and free fatty acid stimulate reactive oxygen species production through pro-

tein kinase C–dependent activation of NAD(P)H oxidase in cultured vascular cells”,

Diabetes, Vol. 49, (2000), pp. 1939–1945.

[12] F. Cosentino, K. Hishikawa, Z.S. Katusic and T.F. Luscher: “High glucose increases

nitric oxide synthase expression and superoxide anion generation in human aortic

endothelial cells”, Circulation, Vol. 96, (1997), pp. 25–28.

[13] M.C. Desco, M. Asensi, R. Marquez, J. Martinez-Valls, M. Vento, F.V. Pallardo, J.

Sastre and J. Vina: “Xanthine oxidase is involved in free radical production in type

1 diabetes: protection by allopurinol”, Diabetes, Vol. 51, (2002), pp. 1118–1124.

[14] L.W. Oberley: “Free radicals and diabetes”, Free Radic. Biol. Med., Vol. 5, (1988),

A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298 297

pp. 113–124.

[15] E.R. Stadtman and C.N. Oliver: “Metal-catalyzed oxidation of proteins. Physiologi-

cal consequences”, J. Biol. Chem., Vol. 266, (1991), pp. 2005–2008.

[16] T. Miyata, R. Inagi, K. Asahi, Y. Yamada, K. Horie, H. Sakai, K. Uchida and

K. Kurokawa: “Generation of protein carbonyls by glycoxidation and lipoxidation

reactions with autoxidation products of ascorbic acid and polyunsaturated fatty

acids”, FEBS Lett., Vol. 437, (1998), pp. 24–28.

[17] T. Grune, T. Reinheckel, M. Joshi and K.J. Davies: “Proteolysis in cultured liver

epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex,

proteasome”, J. Biol. Chem., Vol. 270, (1995), pp. 2344–2351.

[18] T. Reinheckel, N. Sitte, O. Ullrich, U. Kuckelkorn, K.J. Davies and T. Grune: “Com-

parative resistance of the 20S and 26S proteasome to oxidative stress”, Biochem. J.,

Vol. 335, (1998), pp. 637–642.

[19] O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall: “Protein measurement

with the Folin phenol reagent”, J. Biol. Chem., Vol. 193, (1951), pp. 265–275.

[20] F.E. Hunter, Jr., J.M. Gebicki, P.E. Hoffsten, J. Weinstein and A. Scott: “Swelling

and lysis of rat liver mitochondria induced by ferrous ions”, J. Biol. Chem., Vol. 238,

(1963), pp. 828–835.

[21] M.J. Whitekus, N. Li, M. Zhang, M. Wang, M.A. Horwitz, S.K. Nelson, L.D. Hor-

witz, N. Brechun, D. Diaz-Sanchez and A.E. Nel: “Thiol antioxidants inhibit the

adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbu-

min sensitization”, J. Immunol., Vol. 168, (2002), pp. 2560–2567.

[22] A.L. Bulteau, K.C. Lundberg, K.M. Humphries, H.A. Sadek, P.A. Szweda, B. Friguet

and L.I. Szweda: “Oxidative modification and inactivation of the proteasome during

coronary occlusion/reperfusion”, J. Biol. Chem., Vol. 276, (2001), pp. 30057–30063.

[23] D. Voges, P. Zwickl and W. Baumeister: “The 26S proteasome: a molecular machine

designed for controlled proteolysis”, Annu. Rev. Biochem., Vol. 68, (1999), pp. 1015–

1068.

[24] R. Shringarpure, T. Grune and K.J. Davies: “Protein oxidation and 20S proteasome-

dependent proteolysis in mammalian cells”, Cell Mol. Life Sci., Vol. 58, (2001), pp.

1442–1450.

[25] T. Grune, T. Reinheckel, M. Joshi and K.J. Davies: “Proteolysis in cultured liver

epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex,

proteasome”, J. Biol. Chem., Vol. 270, (1995), pp. 2344–2351.

[26] S. Merforth, L. Kuehn, A. Osmers and B. Dahlmann: “Alteration of 20S proteasome-

subtypes and proteasome activator PA28 in skeletal muscle of rat after induction of

diabetes mellitus”, Int. J. Biochem. Cell Biol., Vol. 35, (2003), pp. 740–748.

[27] A.J. Ashford and V.M. Pain: “Effect of diabetes on the rates of synthesis and degra-

dation of ribosomes in rat muscle and liver in vivo”, J. Biol. Chem., Vol. 261, (1986),

pp. 4059–4065.

[28] O.L. Smith, C.Y. Wong and R.A. Gelfand: “Skeletal muscle proteolysis in rats with

acute streptozocin-induced diabetes”, Diabetes, Vol. 38, (1989), pp. 1117–1122.

298 A. Alexandrova et al. / Central European Journal of Biology 1(2) 2006 289–298

[29] H.A. Runnels, W.A. Watkins and J.J. Monaco: “LMP2 expression and proteasome

activity in NOD mice”, Nat. Med., Vol. 6, (2000), pp. 1064–1065.

[30] T. Hayashi and D. Faustman: “NOD mice are defective in proteasome production

and activation of NF-kappaB”, Mol. Cell. Biol., Vol. 19, (1999), pp. 8646–8659.

[31] S.J. Rhee, Y.C. Jeong and J.H. Choi: “Effects of vitamin E on phospholipase A2

activity and oxidative damage to the liver in streptozotocin-induced diabetic rats”,

Ann. Nutr. Metab, Vol. 49, (2005), pp. 392–396.

[32] Y.Y. Jang, J.H. Song, Y.K. Shin, E.S. Han and C.S. Lee: “Protective effect of

boldine on oxidative mitochondrial damage in streptozotocin-induced diabetic rats”,

Pharmacol. Res., Vol. 42, (2000), pp. 361–371.

[33] E. Altomare, I. Grattagliano, G. Vendemaile, T. Micelli-Ferrari, A. Signorile and

L. Cardia: “Oxidative protein damage in human diabetic eye: evidence of a retinal

participation”, Eur. J. Clin. Invest, Vol. 27, (1997), pp. 141–147.

[34] V.M. Bhor, N. Raghuram and S. Sivakami: “Oxidative damage and altered antioxi-

dant enzyme activities in the small intestine of streptozotocin-induced diabetic rats”,

Int. J Biochem. Cell Biol., Vol. 36, (2004), pp. 89–97.

[35] H. Esterbauer, R.J. Schaur and H. Zollner: “Chemistry and biochemistry of 4-

hydroxynonenal, malonaldehyde and related aldehydes”, Free Radic. Biol. Med., Vol.

11, (1991), pp. 81–128.

[36] A. Amici, R.L. Levine, L. Tsai and E.R. Stadtman: “Conversion of amino acid

residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-

catalyzed oxidation reactions”, J. Biol. Chem., Vol. 264, (1989), pp. 3341–3346.

[37] F. Shang, X. Gong and A. Taylor: “Activity of ubiquitin-dependent pathway in re-

sponse to oxidative stress. Ubiquitin-activating enzyme is transiently up-regulated”,

J. Biol. Chem., Vol. 272, (1997), pp. 23086–23093.

[38] M. Portero-Otin, R. Pamplona, M.C. Ruiz, E. Cabiscol, J. Prat and M.J. Bellmunt:

“Diabetes induces an impairment in the proteolytic activity against oxidized proteins

and a heterogeneous effect in nonenzymatic protein modifications in the cytosol of

rat liver and kidney”, Diabetes, Vol. 48, (1999), pp. 2215–2220.

[39] K. Okada, C. Wangpoengtrakul, T. Osawa, S. Toyokuni, K. Tanaka and K. Uchida:

“4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxida-

tive stress. Identification of proteasomes as target molecules”, J. Biol. Chem., Vol.

274, (1999), pp. 23787–23793.

[40] M. Balasubramanyam, R. Sampathkumar and V. Mohan: “Is insulin signaling

molecules misguided in diabetes for ubiquitin-proteasome mediated degradation?”,

Mol. Cell. Biochem., Vol. 275, (2005), pp. 117–125.

[41] M. Kawaguchi, K. Minami, K. Nagashima and S. Seino: “Essential role of ubiquitin-

proteasome system in normal regulation of insulin secretion”, J. Biol. Chem., DOI:

10.1074/jbc.M601228200.