s2fieldsofstudy: Biology Medicine

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Amelioration of galactosamine-induced nephrotoxicity by a protein isolated from the leaves of the herb, Cajanus indicus L

Background Galactosamine (GalN), an established experimental toxin, mainly causes liver injury via the generation of free radicals and depletion of UTP nucleotides. Renal failure is often associated with end stage liver damage. GalN intoxication also induces renal dysfunction in connection with hepatic disorders. Present study was designed to find out the effect of a protein isolated from the leaves of the herb Cajanus indicus against GalN induced renal damage. Methods Both preventive as well as curative effect of the protein was investigated in the study. GalN was administered intraperitoneally at a dose of 800 mg/kg body weight for 3 days pre and post to protein treatment at an intraperitoneal dose of 2 mg/kg body weight for 4 days. The activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione-S-transferase (GST), levels of cellular metabolites, reduced glutathione (GSH), total thiols, oxidized glutathione (GSSG) and lipid peroxidation end products were determined to estimate the status of the antioxidative defense system. In addition, serum creatinine and urea nitrogen (UN) levels were also measured as a marker of nephrotoxicity. Results Results showed that GalN treatment significantly increased the serum creatinine and UN levels compared to the normal group of mice. The extent of lipid peroxidation and the level of GSSG were also enhanced by the GalN intoxication whereas the activities of antioxidant enzymes SOD, CAT, GR and GST as well as the levels of total thiols and GSH were decreased in the kidney tissue homogenates. Protein treatment both prior and post to the toxin administration successfully altered the effects in the experimental mice. Conclusion Our study revealed that GalN caused a severe oxidative insult in the kidney. Protein treatment both pre and post to the GalN intoxication could protect the kidney tissue against GalN induced oxidative stress. As GalN induced severe hepatotoxicity followed by renal failure, the protective role of the protein against GalN induced renal damages is likely to be an indirect effect. Since the protein possess hepatoprotective activity, it may first ameliorate GalN-induced liver damage and consequently the renal disorders are reduced. To the best of our knowledge, this is probably the first report describing GalN-induced oxidative stress in renal damages and the protective role of a plant protein molecule against it.

Background
Galactosamine (GalN) is a well known toxin and has been found to possess various adverse effects in liver. Its toxic effect is associated with the depletion of UTP nucleotides followed by the formation of UDP hexosamines [1] and leads to the inhibition of transcription and consequently the translation processes [2]. The development of acute liver failure due to the administration of GalN is well documented [3][4][5]. Javle et al reported that GalN-induced liver injury is associated with the development of renal failure [6]. Recently, the term hepatorenal syndrome (HRS) has been introduced to define the development of renal failure in the absence of clinical, anatomical or pathological causes of that failure. Classically, HRS is associated with the end-stage liver cirrhosis and it has been observed that renal failure occurs with this liver disease in about 50% of patients [7]. A very recent report of Anand et al revealed that renal failure appeared with acute liver damage where the histological status of the kidney was normal but the renal blood flow was decreased [8].
Although oxidative stress has been reported as one of the major causes in GalN induced liver damages [9][10][11], it is not, however, clear whether GalN-induced renal disorders occur via the generation of reactive oxygen species followed by oxidative stress in the kidney or not.
Herbal medicines have been used traditionally world wide for the prevention and treatment of various diseases. Earlier studies reported that medicinal herbs play a protective role against GalN induced liver damage [12][13][14]. In India medicinal plants are widely used in the ayurvedic medicines for the treatment of various health problems. Some of the important medicinal plants are Silybum marianum [15,16]; Picrorhiza kurroa [17]; Curcuma longa [18]; Phyllanthus niruri [19][20][21][22][23]; Terminalia arjuna [24][25][26]; Andrographis paniculata [27] etc. The herb Cajanus indicus, an important medicinal plant, has been widely used in folklore medicine in India and many other countries for the treatment of various hepatic disorders [28,29]. In our laboratory a 43 kD protein has been isolated and purified to homogeneity from the leaves of this herb [30] and believed to be an active principle responsible for its beneficial action. It has been found that that the protein possesses a protective role against various toxins (such as carbon tetrachloride, chloroform, thioaectamide etc.) induced hepatic disorders [31][32][33][34]. The free radical scavenging activity of this protein was determined from its DPPH radical quenching ability [31]. The present study was conducted to find out whether GalN caused renal damage by inducing oxidative stress in the kidney and whether the protein of our interest could ameliorate that effect. Experiments were designed accordingly to evaluate both the preventive as well as curative role of this protein against GalN induced renal toxicity in vivo. The extent of renal damage caused by GalN intoxication and the protec-tive effect of the protein were evaluated by measuring the i) level of serum creatinine and urea nitrogen (UN) ii) activities of the intracellular enzymes namely, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione-S-transferase (GST) iii) levels of cellular metabolites like reduced glutathione (GSH), total thiols, oxidized glutathione (GSSG) and iv) extent of lipid peroxidation in the kidney tissue homogenates of all the experimental animals.

Plant
Cajanus indicus is a shrub belonging to the family Leguminosae and subfamily papilionacea. Fresh young leaves were collected from Bose Institute experimental farm.

Animals
Swiss albino mice (male, body weight 20 ± 2 g) were used for the experiments. The animals were acclimatized under standard laboratory conditions for a fortnight before starting the experiments. They were provided with standard diet and water ad libitum. They were maintained under standard conditions of temperature (30°C) and humidity (50%) with an alternating 12 hours light/dark cycles. All the experimental studies were conducted in conformity with the guidance for care and standard experimental animals study ethical protocols. The animals were divided into several groups, each group having six mice.

Preparation of the homogeneous protein from the leaves of Cajanus indicus
The protein was purified from the leaves of Cajanus indicus according to the method described by Sarkar et al [30]. The leaves were homogenized in 20 mM tris-HCl buffer, pH 7.4 and the supernatant was brought to 60% (NH 4 ) 2 SO 4 saturation. The pellet was reconstituted and dialyzed in tris-HCl buffer, passed through DEAE Sepha-dex column and eluted in linear gradient of 0-1 M NaCl in tris buffer. The active fraction eluted at 0.2 M NaCl was concentrated and applied on a Sephadex G-50 column. The bioactive fraction obtained was subjected to a C 18 hydrophobic column for reverse phase column chromatography. The homogeneity of preparation was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein estimation
The protein concentration of the experimental samples was measured according to the method of Bradford [35] using crystalline BSA as standard.

Pre treatment with the protein
The pre-treatment group was divided into five sub-groups each consisted of six mice. The first group, which received only the vehicle, served as normal control. The second group, served as toxin control, in which GalN was administered intraperitoneally at a dose of 800 mg/kg body weight once daily and the treatment was carried out for 3 days. The third group of mice was pre-treated with the protein by intraperitoneal injection at a dose of 2 mg/kg body weight once daily and the treatment was carried out for 4 days followed by the toxin treatment for another 3 days. The animals were then sacrificed under anesthesia and kidneys were collected. A well-known antioxidant agent vitamin E, (at a dose of 200 mg/kg body weight) and a non-relevant protein, BSA (at a dose of 2 mg/kg body weight) were used as the positive and negative controls respectively for the study.

Post-treatment with the protein
The post-treatment group was divided into five subgroups each consisted of six mice. The first group, served as normal control, received only the vehicle. The second group received GalN at a dose of 800 mg/kg body weight intraperitoneally (once daily) for 3 days and served as the toxin control. The third group of mice was intoxicated with GalN (800 mg/kg body weight, once daily) for 3 days followed by intraperitoneal administration of the protein at a dose of 2 mg/kg body weight once daily for 4 days. The fourth group received GalN (800 mg/kg body weight, once daily) for 3 days and then kept for another 4 days without any treatment to observe whether there is any natural recovery or not. A positive control group was kept in which mice were treated with 200 mg/kg body weight vitamin E for 4 days after GalN intoxication. Mice were then sacrificed under anesthesia and kidneys were collected.

Preparation of kidney homogenates
The kidney tissues were homogenized using glass homogenizer in 100 mM potassium phosphate buffer containing 1 mM EDTA, pH 7.4 and centrifuged at 12,000 g for 30 minutes at 4°C. The supernatant was collected and used for following experiments.

Assessment of marker of nephrotoxicity
Estimation of creatinine level Blood samples collected from puncturing mice hearts were kept overnight at 4°C to clot and then centrifuged at 3,000 g for 10 minutes. Creatinine level was measured from serum samples according to the method of Bonses and Taussky [36].

Estimation of UN level
Estimation of UN level has been carried following the method of Crocker [37]. Like creatine measurement, blood samples were collected from puncturing mice hearts and kept overnight at 4°C to clot. Sera from the samples were collected by centrifuging the blood samples at 3,000 g for 10 minutes. UN level in all the experimental samples were measured by using a kit from Span Diagnostics Limited, India.

Estimation of lipid peroxidation end products
The extent of lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) formation was measured according to the method of Esterbauer and Cheeseman [38] using the extinction co-efficient of MDA, which is 1.56 × 10 5 M -1 cm -1 .

Assay of antioxidant enzymes SOD assay
The activity of SOD was measured following the method originally developed by Nishikimi [39] and then modified by Kakkar [40]. One unit of SOD activity is defined, as the enzyme concentration required inhibiting chromogen production by 50% in one minute under the assay condition.

CAT assay
The enzyme CAT converts H 2 O 2 into water. The CAT activity was measured by the method of Bonaventura [41]. One unit of CAT activity is defined as the amount of enzyme, which reduces 1 µmol of H 2 O 2 per minute.
GST assay GST catalyzes the conjugation reaction with glutathione in the first step of mercapturic acid synthesis. The activity of GST was measured by the method of Habig and Jakoby [42]. One unit of GST activity is 1 µmol product formation per minute.
Assay of GR GR activity was measured according to the method of Smith [43]. The enzyme activity was calculated using molar extinction coefficient of 13,600 M -1 cm -1 . One unit of enzyme activity is defined as the amount of enzyme, which catalyzes the oxidation of 1µmol NADPH per minute.

Assay of cellular metabolites from kidney homogenates
GSH assay GSH level was measured according to the method of Ellman [44]. A standard curve was drawn using different known concentrations of GSH solution. With the help of this standard curve, GSH contents were calculated.
GSSG assay GSSG level was measured according to the method of Hissin and Hilf [45]. The absorbance of the sample was measured at 412 nm.

Assay of total thiols
The total thiols (total sulfhydryl groups) content was measured according to the method of Sedlak and Lindsay [46] with some modifications. The content of total thiols was calculated using molar extinction coefficient of 13,600 M -1 cm -1 .

Statistical analysis
All the values are represented as mean ± S.D. (n = 6). The statistical differences among different groups were analised by Student's t-test. P values of 0.05 or less were considered significant.

Effect on nephrotoxicity markers Creatinine levels
The creatinine levels in serum obtained from the normal control mice, toxin control mice, mice treated separately with protein, vitamin E and BSA followed by GalN intoxication, mice treated separately with protein, vitamin E after GalN intoxication and mice kept for normal recovery after GalN administration are shown in figure 1. The creatinine level was elevated in toxin control (0.85 ± 0.038 mg/dl) compared to the normal control (0.66 ± 0.026 mg/dl), whereas protein pre-treatment reduced the level (0.68 ± 0.029 mg/dl). There was reduction of creatinine level (0.65 ± 0.033 mg/dl) in protein post-treated group of mice. The normal recovery after toxin administration was very slow (0.79 ± 0.036 mg/dl). Figure 2 represents UN level in serum of all the experimental animals. In toxin control an increase in UN level (44.68 ± 1.98 mg/dl) has been observed compared to the normal control (26.57 ± 1.22 mg/dl), whereas protein pre-treatment attenuated the level (30.71 ± 1.39 mg/dl). Protein post treatment also reduced the UN level (27.47 ± 1.29 mg/dl). The normal recovery after toxin administration was not significant (40.62 ± 1.78 mg/dl). Figure 3 shows the levels of MDA in kidney tissue homogenates obtained from all the experimental mice. MDA level (41.22 ± 0.92 nmol/g tissue) in kidney tissue homogenates of GalN treated mice was found to be higher than that level compared to normal control mice (27.99 ± 0.59 nmol/g tissue). Pre-treatment with the protein followed by toxin administration decreased the level (15.35 ± 0.45 nmol/g tissue). Post treatment with the protein after toxin administration decreased MDA level (10.17 ± 0.52 nmol/g tissue) also. The normal recovery after toxin treatment was very slow (40.91 ± 0.82 nmol/g tissue).

Effect on the end products of lipid peroxidation
Effect on antioxidant enzymes SOD activities SOD activities in kidney tissue homogenates of all experimental mice are shown in figure 4. SOD activity was reduced in toxin control mice (19.21 ± 0.85 unit/mg protein) compared to the normal control (80.55 ± 1.61 unit/ mg protein). There was increase in SOD activity in the protein treated mice (85.65 ± 1.71 unit/mg protein) prior to GalN administration and in protein treated mice (99.82 ± 2.09 unit/mg protein) after GalN administration. No significant normal recovery (30.25 ± 0.83 unit/mg protein) after toxin treatment was observed.
CAT activities CAT activities in kidney tissue homogenates of all experimental mice are shown in figure 5. There was decreased CAT activity in toxin control (176.96 ± 3.89 µmol/min/ mg protein) compared to the normal control (348.31 ± 6.62 µmol/min/mg protein). Pre as well as post-treatment with the protein increased CAT activity. The activities were 336.06 ± 7.22 µmol/min/mg protein and 344.49 ± 7.41 µmol/min/mg protein respectively. The normal recovery after toxin treatment was not significant (192.95 ± 4.51 µmol/min/mg protein).
GST activities GST activities in kidney tissue homogenates of all experimental mice are shown in figure 6. A decreased GST activity was observed in toxin control (1.16 ± 0.03 µmol/min/ mg protein) compared to the normal control (3.01 ± 0.08 µmol/min/mg protein). Protein treatment prior to GalN intoxication caused an elevation in GST activity (3.35 ± 0.09 µmol/min/mg protein). GST activity (2.78 ± 0.04 µmol/min/mg protein) was also elevated in mice group treated with protein after toxin administration. The normal recovery after toxin treatment was very slow (1.48 ± 0.03 µmol/min/mg protein).
GR activities GR activities in kidney tissue homogenates obtained from all the experimental mice are shown in figure 7. There was reduction of GR activity in toxin control (142.18 ± 2.74 Effect of the protein on creatinine level in GalN induced kidney damage in mice Figure 1 Effect of the protein on creatinine level in GalN induced kidney damage in mice. Left and right panel show preventive and curative effect of the protein respectively. Cont: creatinine level in normal mice, GalN: creatinine level in only GalN treated mice, Protein + GalN: creatinine level in which the protein was given prior to GalN administration, VitE + GalN: creatinine level in which vitamin E was administered prior to GalN treatment, BSA + GalN: creatinine level in which BSA was given before GalN administration. GalN + Protein: creatinine level in which the protein was given after GalN administration, GalN + Vit E: creatinine level in which vitamin E was administered post to GalN treatment and Recovery: creatinine level in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). UN level in which the protein was given prior to GalN administration, VitE + GalN: UN level in which vitamin E was administered prior to GalN treatment, BSA + GalN: UN level in which BSA was given before GalN administration. GalN + Protein: UN level in which the protein was given after GalN administration, GalN + Vit E: UN level in which vitamin E was administered post to GalN treatment and Recovery: UN level in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). GalN + Protein: MDA content in which the protein was given after GalN administration, GalN + Vit E: MDA level in which vitamin E was administered post to GalN treatment and Recovery: MDA content in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). Effect of the protein on SOD activity in GalN induced kidney damage in mice. Left and right panel show preventive and curative effect of the protein respectively. Cont: SOD activity in normal mice, GalN: SOD activity in only GalN treated mice, Protein + GalN: SOD activity in which the protein was given prior to GalN administration, VitE + GalN: SOD activity in which vitamin E was administered prior to GalN treatment, BSA + GalN: SOD activity in which BSA was given before GalN administration. GalN + Protein: SOD activity in which the protein was given after GalN administration, GalN + Vit E: SOD activity in which vitamin E was administered post to GalN treatment and Recovery: SOD activity in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). Effect of the protein on CAT activity in GalN induced kidney damage in mice. Left and right panel show preventive and curative effect of the protein respectively. Cont: CAT activity in normal mice, GalN: CAT activity in only GalN treated mice, Protein + GalN: CAT activity in which the protein was given prior to GalN administration, VitE + GalN: CAT activity in which vitamin E was administered prior to GalN treatment, BSA + GalN: CAT activity in which BSA was given before GalN administration. GalN + Protein: CAT activity in which the protein was given after GalN administration, GalN + Vit E: CAT activity in which vitamin E was administered post to GalN treatment and Recovery: CAT activity in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). Effect of the protein on GST activity in GalN induced kidney damage in mice. Left and right panel show preventive and curative effect of the protein respectively. Cont: GST activity in normal mice, GalN: GST activity in only GalN treated mice, Protein + GalN: GST activity in which the protein was given prior to GalN administration, VitE + GalN: GST activity in which vitamin E was administered prior to GalN treatment, BSA + GalN: GST activity in which BSA was given before GalN administration. GalN + Protein: GST activity in which the protein was given after GalN administration, GalN + Vit E: GST activity in which vitamin E was administered post to GalN treatment and Recovery: GST activity in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). nmol/min/mg protein) compared to the normal control (293.38 ± 6.78 nmol/min/mg protein). Elevation of GR activity (340.12 ± 8.33 nmol/min/mg protein) was observed in protein pre-treatment group. Post treatment with the protein increased GR activity (303.82 ± 7.17 nmol/min/mg protein) also. A little normal recovery (166.72 ± 4.05 nmol/min/mg protein) after toxin treatment was observed. Figure 8 shows the levels of GSH in kidney tissue homogenates obtained from all the experimental mice. There was decrease of GSH level in toxin control (33.77 ± 1.52 nmol/mg protein) compared to that of normal control (48.41 ± 2.28 nmol/mg protein), whereas protein pretreatment increased the level (50.44 ± 2.32 nmol/mg protein). An increased GSH level (51.23 ± 2.43 nmol/mg protein) was observed in kidney homogenates obtained from the mice treated with the protein after toxin administration. There was no significant normal recovery (37.01 ± 1.77 nmol/mg protein) after toxin treatment.

GSSG levels
The levels of GSSG in kidney tissue homogenates obtained from all the experimental mice are shown in figure 9. GSSG level in toxin control (2.24 ± 0.098 nmol/mg protein) was found to be higher than that level compared to normal control (1.14 ± 0.051 nmol/mg protein). Pretreatment with the protein followed by toxin administration decreased the level (1.11 ± 0.049 nmol/mg protein). GSSG level (1.09 ± 0.052 nmol/mg protein) has been found to decrease in post-treatment group of mice. The normal recovery after toxin treatment was very slow (2.01 ± 0.095 nmol/mg protein). Figure 10 shows the levels of total thiols in kidney tissue homogenates obtained from all the experimental mice groups. Toxin treatment decreased the level of total thiols (253.28 ± 5.32 nmol/mg protein) compared to the normal control group (541.25 ± 10.83 nmol/mg protein) whereas protein pre-treatment increased the level (440.83 ± 11.25 nmol/mg protein). Post treatment of the protein after GalN intoxication increased the level of total thiols (433.73 ± 10.11 nmol/mg protein) compared to toxin control group. No significant normal recovery (264.88 ± 5.29 nmol/mg protein) after toxin treatment was found.

Levels of total thiols
Vitamin E treatment pre and post to GalN intoxication could protect kidney by preventing alterations in all the antioxidant indices described in the present study, whereas BSA did not show any protective activity.

Discussion
The aim of the present study was to investigate whether the protein isolated from the herb Cajanus indicus possesses any preventive as well as curative role against GalN induced renal damages. Results suggest that administration of GalN at a dose of 800 mg/kg body weight for 3 days significantly altered the status of the antioxidant defense system in the kidney. Protein treatment both pre and post to the toxin administration restored the activities of all the intracellular antioxidant enzymes as well as level of cellular metabolites.
Reports on the mechanism of GalN induced hepatotoxicity suggests that GalN inhibits the synthesis of RNA as well as protein via reduction of cellular UTP, a substrate for RNA polymerase. GalN shifts the equilibrium between the uridine phosphate consuming and producing reactions, in favor of the former via the rapid formation and accumulation of UDP amino sugars [2]. Studies from Quintero et al [47] showed that free radical dependent pathway is involved in GalN induced hepatic apoptosis and necrosis in rat hepatocytes. Mangeney-Andreani et al [10] suggested that energy metabolism of hepatocytes was also inhibited by GalN application. In addition, report from Sire et al [48] reveals that GalN damaged the enzymes involved in the transport of substrates to the mitochondria and consequently modified the phospholipid composition of the membrane. It has been observed that patient suffering with liver disease have a decrease in renal blood flow, indicating renal vasoconstriction and this effect is more prominent in those people affected with HRS [49]. Earlier studies reported that endothelin-1 (ET-1), one of the most potent vasoconstrictor in biological system, induces renal vasoconstriction as well as reduction in glomerular filtration rate (GFR), renal blood flow and sodium excretion [50]. Uchida et al [51] and Sakamoto et al [52] reported that various cells within the kidney tissue either express mRNA for ET-1 or can synthesis ET-1 and thus it reveals that ET-1 may, therefore, have a paracrine or autocrine effect. It has been observed that the levels of plasma ET-1 concentration are elevated in patients with severe liver disease and HRS [53]. Annand et al [8] reported that GalN intoxication causes liver injury followed by renal impairment, which is manifested by the decrease in renal blood flow, creatinine clearance as well as increase in plasma ET-1 concentration and also up-regulation of ET-1 receptor in renal cortex although kidneys are histologically normal. Thus study also suggests that renal failure also appears to be secondary to liver failure.
In the present study we found that GalN administration at a dose of 800 mg/kg body weight for three days caused enhanced oxidative insult in the kidney tissue as evidenced by the increased renal TBARS level and depletion in the activities of endogenous antioxidant enzymes such Effect of the protein on GR activity in GalN induced kidney damage in mice Figure 7 Effect of the protein on GR activity in GalN induced kidney damage in mice. Left and right panel show preventive and curative effect of the protein respectively. Cont: GR activity in normal mice, GalN: GR activity in only GalN treated mice, Protein + GalN: GR activity in which the protein was given prior to GalN administration, VitE + GalN: GR activity in which vitamin E was administered prior to GalN treatment, BSA + GalN: GR activity in which BSA was given before GalN administration. GalN + Protein: GR activity in which the protein was given after GalN administration, GalN + Vit E: GR activity in which vitamin E was administered post to GalN treatment and Recovery: GR activity in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). GSH level in which the protein was given prior to GalN administration, VitE + GalN: GSH level in which vitamin E was administered prior to GalN treatment, BSA + GalN: GSH level in which BSA was given before GalN administration. GalN + Protein: GSH level in which the protein was given after GalN administration, GalN + Vit E: GSH level in which vitamin E was administered post to GalN treatment and Recovery: GSH level in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). Effect of the protein on GSSG level in GalN induced kidney damage in mice. Left and right panel show preventive and curative effect of the protein respectively. Cont: GSSG level in normal mice, GalN: GSSG level in only GalN treated mice, Protein + GalN: GSSG level in which the protein was given prior to GalN administration, VitE + GalN: GSSG level in which vitamin E was administered prior to GalN treatment, BSA + GalN: GSSG level in which BSA was given before GalN administration. GalN + Protein: GSSG level in which the protein was given after GalN administration, GalN + Vit E: GSSG level in which vitamin E was administered post to GalN treatment and Recovery: GSSG level in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). Effect of the protein on levels of total thiols in GalN induced kidney damage in mice Figure 10 Effect of the protein on levels of total thiols in GalN induced kidney damage in mice. Left and right panel show preventive and curative effect of the protein respectively. Cont: total thiol level in normal mice, GalN: total thiol level in only GalN treated mice, Protein + GalN: total thiol level in which the protein was given prior to GalN administration, VitE + GalN: total thiol level in which vitamin E was administered prior to GalN treatment, BSA + GalN: total thiol level in which BSA was given before GalN administration. GalN + Protein: total thiol level in which the protein was given after GalN administration, GalN + Vit E: total thiol level in which vitamin E was administered post to GalN treatment and Recovery: total thiol level in mice which were treated with GalN for 3 days and kept next 4 days without any treatment. Each column represents mean ± SD, n = 6. "a" indicates the significant difference between the vehicle control and toxin treated groups, "b" indicates the significant difference between the toxin treated and protein treated groups and "c" indicates the significant difference between the toxin treated and vitamin E treated groups. (P a < 0.01, P b < 0.01, P c < 0.01). as SOD, CAT, GR and GST. GalN intoxication also reduced the level of total thiol and GSH followed by the increase in GSSG level. The renal dysfunction due to GalN treatment was also manifested by the increase in serum creatinine and UN levels as compared to the normal group of mice. Protein treatment at a dose of 2 mg/kg body weight for 4 days both prior and post to the toxin administration attenuated the increased creatinine and UN levels compared to the toxin control. Administration of the protein restored the activities of intracellular antioxidant enzymes SOD, CAT, GR and GST. The decreased level of ROS scavengers like GSH and total thiols were also enhanced by the protein treatment both prior and post to the GalN intoxication. The content of total thiols includes the protein-bound thiols as well as nonprotein-bound thiols. It has been reported that the former usually contribute more (a few fold higher than the later) to the total thiols contents [54]. The protein also ameliorated the enhanced level of TBARS as well as the level of GSSG compared to the toxin control.
As reported from the earlier studies of our laboratory, the protein could scavenge DPPH radicals as well as restore the activities of different antioxidant enzymes and attenuate various toxin-induced enhancement of lipid peroxidation [31][32][33][34]. The detail mechanism of action of this protein necessary for the protective action against hepatic and renal damages is not yet fully known. As it possesses hepatoprotective activity, it appears that the amelioration of renal damage may not be its direct action to the kidney. GalN induced renal failure appears at the end stage of liver cirrhosis; so protecting the liver from that severe damage it indirectly possesses protective role against GalNinduced disorders in the kidney.
Although a number of very recent reports revealed that protein molecules from various plant sources possess antioxidant and hepatoprotective activities like the protein of our interest [55][56][57], similarities among their structural features could not be related at this stage because of the lack of knowledge on their structures. These reports also lack the knowledge of the exact mechanism of actions of those protein molecules; only some biochemical properties are known and that could be compared to the protein of our interest.

Conclusion
In conclusion we would like to mention that GalN administration caused oxidative insult in the kidney. Protein treatment both prior and post to the toxin administration normalize that stress in the organ. As GalN induced renal failure seems to occur at the end stage of liver cirrhosis, the protective role of the protein against GalN induced renal damages is likely to be an indirect effect probably comes to play via the protection of hepatic disorders. Further works are however needed to define the exact mechanisms by which the protein exhibits this protective action and are currently in progress.


extfieldsofstudy: Biology Medicine

text:
Proteomics Analysis of Insulin Secretory Granules*S

Insulin secretory granules (ISGs) are cytoplasmic organelles of pancreatic β-cells. They are responsible for the storage and secretion of insulin. To date, only about 30 different proteins have been clearly described to be associated with these organelles. However, data from two-dimensional gel electrophoresis analyses suggested that almost 150 different polypeptides might be present within ISGs. The elucidation of the identity and function of the ISG proteins by proteomics strategies would be of considerable help to further understand some of the underlying mechanisms implicated in ISG biogenesis and trafficking. Furthermore it should give the bases to the comprehension of impaired insulin secretion observed during diabetes. A proteomics analysis of an enriched insulin granule fraction from the rat insulin-secreting cell line INS-1E was performed. The efficacy of the fractionation procedure was assessed by Western blot and electron microscopy. Proteins of the ISG fraction were separated by SDS-PAGE, excised from consecutive gel slices, and tryptically digested. Peptides were analyzed by nano-LC-ESI-MS/MS. This strategy identified 130 different proteins that were classified into four structural groups including intravesicular proteins, membrane proteins, novel proteins, and other proteins. Confocal microscopy analysis demonstrated the association of Rab37 and VAMP8 with ISGs in INS-1E cells. In conclusion, the present study identified 130 proteins from which 110 are new proteins associated with ISGs. The elucidation of their role will further help in the understanding of the mechanisms governing impaired insulin secretion during diabetes.

Insulin is a key molecule for the organism because it regulates glucose homeostasis (1), lipid and protein metabolism (2, 3), brain function (4), and cell survival (5). Pancreatic ␤-cells are the functional units responsible for the storage and secretion of insulin (6). These cells are able to adapt their rate of insulin secretion to blood glucose variations (7). Insulin is initially translated as a proinsulin precursor in the endoplasmic reticulum (ER), 1 is then transported from the ER to the Golgi apparatus, and crosses the Golgi network before being sorted into clathrin-coated immature insulin secretory granules (ISGs). The acidification of the lumen of immature ISGs through the action of ATPase proton pumps coincides with the cleavage of proinsulin into insulin and peptide C (8 -10). These biochemical modifications trigger the maturation to mature ISGs. After glucose stimulation, mature ISGs release their insulin content into the extracellular space by exocytosis (11). One primary ␤-cell contains around 10,000 ISGs; however, only 100 -200 ISGs are capable to quickly release their insulin content in response to an increased intracellular [Ca 2ϩ ] (12). The majority of ISGs belong to a reserve pool and must undergo several ATP-dependent reactions to become competent for final exocytosis (11,13). Suppression of ISG-associated proteins such as vesicle-associated membrane protein 2 (VAMP2) or Rab3a affects insulin secretion demonstrating the functional importance of these proteins in the regulated insulin release (14,15).
Whether insulin resistance or insulin secretion defects are primary in the development of type 2 diabetes has been a highly debated topic, and there is no consensus. However, it is clear that overt type 2 diabetes only occurs when the insulin output from the pancreatic islets fails to match the insulin requirement as a result of the insulin resistance (16). It is not clear whether the primary decline in insulin secretion results from a reduction in the number of ␤-cells, a progressive dysfunction of some of the ␤-cells, or a combination of both (17). The molecular mechanisms leading to insulin secretion impairment may partly be due to the modulation of the expression of several key ISG proteins. Their identification would provide a better picture of the events involved in the onset of diabetes. So far only about 30 proteins have been clearly described to be associated with ISGs. However, according to 2-DE gel analysis, Hutton (18) suggested that ISGs potentially contain more than 150 polypeptides. To date the identity of these proteins remains to be determined.
This work presents for the first time a comprehensive picture of the ISG proteome from the rat ␤-cell line INS-1E. This cell line has previously proven useful for the elucidation of secretory granule protein function (19,20). The ISG fraction was obtained by a two-step subcellular fractionation strategy, its protein content was separated by SDS-PAGE, and 130 proteins were identified by tandem mass spectrometry. Finally immunofluorescence experiments showed the newly found presence of Rab37 and VAMP8 proteins on ISGs.

EXPERIMENTAL PROCEDURES
Materials-All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich and were of the highest purity available. CH 3 CN was purchased from Biosolve (Westford, MA). Percoll solution was obtained from GE Healthcare. Rabbit polyclonal antibody against betagranin was from Eurogentech (Seraing, Belgium), mouse monoclonal antibody against cathepsin L and pig polyclonal antibody against insulin were from Abcam (Cambridge, UK), rabbit polyclonal antibody against VAMP8 was from Synaptic System (Gottingen, Germany), mouse monoclonal antibody against GS-28 was from Calbiochem (EMB Biosciences), rabbit polyclonal antibody against lysosome-associated membrane protein 2 (Lamp2) was from Zymed Laboratories Inc.(Invitrogen), rabbit polyclonal antibody against cathepsin B was from Upstate (Chicago, IL), rabbit polyclonal antibody against calreticulin was a gift from Dr. K. H. Krause (University of Geneva, Geneva, Switzerland), rabbit polyclonal antibody against Glut2 was a gift from Dr. P. Meda (University of Geneva, Geneva, Switzerland), and rabbit polyclonal antibodies against Rab5 and cathepsin D were a gift from Dr. J. Gruenberg (University of Geneva, Geneva, Switzerland). Fluorescent dye-conjugated secondary antibodies (546nm anti-rabbit, 546nm anti-mouse, 488nm anti-rabbit, 488nm anti-mouse, and 488nm anti-pig) were from Fluoprobes (Chemie-Brunschwig, Basel, Switzerland). Insulin content was measured by ELISA (Mercodia, Uppsala, Sweden) according to the manufacturer's instructions.
Cell Culture and Transfection-The INS-1E clone from the insulinsecreting cell line INS-1 was cultured in RPMI 1640 medium and other additions as described by Merglen et al. (21). INS-1E cells were transfected with Lipofectamine TM 2000 (Invitrogen) according to the manufacturer's instructions. The mouse Rab37 cDNA (GenBank TM accession number AB232633) was amplified by PCR and subcloned into the BgIII/EcoRI site of pEGFP-C1 vector (Clontech) as described previously (22). Subcellular Fractionation for Insulin Secretory Granules Purification-ISGs were obtained by subcellular fractionation from INS-1E cells. All procedures were performed at 4°C. Briefly about 8 ϫ 10 8 INS-1E cells grown in 600-cm 2 dishes were washed once with PBS and scraped in cold PBS. Cells were then homogenized in 15 ml of 0.27 M sucrose, 10 mM MOPS-Tris (pH 6.8) (SMT) by three strokes through a 21-gauge needle followed by three strokes through a 25-gauge needle. Cell debris and nuclei were removed by centrifugation for 5 min at 1000 ϫ g. The supernatant was transferred to a new tube, and the remaining pellet was homogenized in 15 ml of SMT by three strokes through a 21-gauge needle followed by five strokes through a 25-gauge needle. The homogenate was centrifuged for 5 min at 1000 ϫ g, and the resulting supernatant was pooled with the first one. The pooled supernatant was finally centrifuged for 10 min at 1000 ϫ g to obtain the postnuclear supernatant (PNS). The PNS was then centrifuged at 24,700 ϫ g for 20 min to separate organelles from the cytosol. The resulting pellet was resuspended in SMT, loaded on a discontinuous Nycodenz gradient composed of three layers (23.4, 8.8, and 4.4%) and centrifuged at 107,000 ϫ g for 75 min. The Nycodenz enriched insulin granule fraction was recovered and loaded on a 27% Percoll solution, which was centrifuged at 35,000 ϫ g for 45 min. The Percoll enriched insulin granule fraction was recovered and washed three times with SMT to eliminate colloidal particles of Percoll.
Transmission Electron Microscopy-Fixation of the ISG pellet was performed by incubation in 2.5% glutaraldehyde for 1 h. The fixed pellet was washed three times with PBS, dehydrated, embedded in epoxy resin, and processed for electron microscopy as described previously (23). Ultrathin sections were contrasted with uranyl acetate and lead citrate and observed with a Technai 20 (FEI Co., Eindhoven, Netherlands) electron microscope.
Protein Separation by SDS-PAGE-Prior to SDS-PAGE, proteins were quantified using a Bradford assay (Bio-Rad). Equal amounts of proteins from the total cells and from the different cellular fractions were solubilized in sample buffer (62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol, 0.1 M dithiothreitol, and traces of bromphenol blue) before separation on a 12.5% T, 2.6% C polyacrylamide gel (71). After electrophoresis proteins were stained with Coomassie Blue R-250 (Merck).
Immunoblotting-Seven micrograms of protein from each cellular fraction was separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Bio-Rad). Immunoreactive bands were revealed by enhanced chemiluminescence using horseradish peroxidase-coupled secondary antibodies (1:2000) (Roche Applied Science).
Protein Identification by LC-MS/MS-A 1-DE gel lane corresponding to insulin secretory granules was cut in 28 consecutive pieces of gel. For each piece of gel "in-gel" digestion was performed as described by Scherl et al. (24). LC-MS/MS analysis with the LCQ ion trap (Thermo Finnigan, San Jose, CA) was performed as described by Burgess et al. (25). Peak lists were generated using Bioworks 3.1 software (Thermo Finnigan). The resulting .dta files from each analysis were automatically combined into a single text file. The resulting peak lists were searched against the rat International Protein Index (IPI) database version 3.20 (41,546 entries, European Bioinformatics Group) using Mascot operating on a local server (version 2.1, Matrix Science) and Phenyx (GeneBio). Mascot was used with average mass selected, a precursor mass error of 2.0 Da, and a peptide mass error of 1.0 Da. Trypsin was selected as the enzyme with three potential missed cleavages. ESI ion trap was selected as the instrument type, and oxidized methionine and carbamidomethylated cysteine were selected as variable modifications. For Phenyx, ion trap was selected for the instrument type, and LCQ was selected for the algorithm. Two search rounds were used, both with trypsin selected as the enzyme and oxidized methionine and carbamidomethylated cysteine selected as variable modifications. In the first round one missed cleavage was allowed, and the normal cleavage mode was used. This round was selected in "turbo" search mode. In the second round three missed cleavages were allowed, and the cleavage mode was set to halfcleaved. The minimum peptide length allowed was 6 amino acids, and the parent ion tolerance was 2.0 Da in both search rounds. The acceptance criteria were slightly lowered in the second round search (round 1: AC score, 7.0; peptide Z-score, 7.0; peptide p value, 1EϪ7; round 2: AC score, 7.0; peptide Z-score, 6.0; peptide p value, 1EϪ6). Only proteins that were identified with one or more high scoring peptides from both Mascot and Phenyx were considered to be true matches. "High scoring peptides" corresponded to peptides that were above the threshold in Mascot (p Ͻ 0.05) and Phenyx (p value Ͼ1EϪ7) searches. Peptides common to different proteins are in italic in Supplemental Table 2. All identified proteins were manually confirmed to contain at least one specific and non-redundant peptide.
Immunocytochemistry-INS-1E cells cultured on coverslips were fixed for 20 min in 3% (w/v) paraformaldehyde in PBS, washed three times with PBS, and exposed for 20 min in 50 mM NH 4 Cl to avoid autofluorescence. Cells were then washed three times with PBS, blocked for 15 min in 10% FCS, and exposed for 45 min in PBS containing primary antibodies, 0.1% saponin, and 10% FCS. Cells were then exposed to fluorescent dye-conjugated secondary antibodies (1:100) for 30 min at room temperature. Samples were analyzed using a Zeiss laser confocal microscope (LSM 510 Meta, Carl Zeiss AG, Zurich, Switzerland). Images were taken with a 60ϫ objective.

RESULTS
Efficiency of the Subcellular Fractionation-Efficient organelle proteomics analysis always depends on the quality of subcellular fractionation and organelle enrichment. Here the common continuous sucrose density gradient (0.45-2 M) permitted the separation of the two vesicular populations in INS-1 cells: ISGs and synaptic-like microvesicles (26). However, Western blot analyses of the obtained ISG fractions revealed the presence of calreticulin, indicating an ER contamination (data not shown). Therefore, we established a two-step subcellular fractionation protocol, which is composed of an initial discontinuous Nycodenz gradient followed by a 27% Percoll solution (Fig. 1A). To determine the enriched ISG fraction in the Nycodenz gradient, insulin was quantified in 12 fractions. Fraction 8 exhibited the largest level of insulin (Fig. 1B, left panel). Western blotting performed with antibodies against betagranin (N-terminal fragment of chromogranin A) or calreticulin (ER-resident protein) showed that fraction 8 contains higher levels of betagranin and lower levels of calreticulin (Fig. 1B, right panel). Thus, fraction 8 was chosen for subsequent separation on a Percoll solution. Analysis of the different fractions showed that in fraction 12 of this Percoll solution calreticulin was absent, whereas insulin and betagranin were present in high amounts (Fig. 1C). This fraction, referred to as the ISG fraction, was selected for subsequent proteomics analysis.
Subcellular Fractionation Validation by 1-DE Gels, Immunoblotting, and Electron Microscopy-To evaluate the ISG en-richment, 25 g of proteins from each cellular fraction was separated by SDS-PAGE and stained with Coomassie Blue (Fig. 2A). The band patterns obtained from total cells, PNS, and cytoplasmic fractions were very similar. However, the band pattern of the ISG fraction was clearly different. Similar results were observed by silver staining (data not shown). These data demonstrate that this fraction is highly enriched in specific proteins.
Consistent with these data, electron microscopy analysis revealed that the ISG fraction was highly enriched in ISGs. Furthermore ISGs maintained an intact morphology with the typical insulin dense core surrounded by filamentous structures ( Fig. 2C) (27).
Immunocytochemistry Validation-Because numerous lysosomal hydrolases were identified in the C1 class, we first evaluated the cellular distribution of cathepsin B, D, and L by immunofluorescence in INS-1E cells (Fig. 5). Confocal microscopy images showed that all three isoforms are expressed in compartments containing immunoreactive insulin. These pro-teins were also detected in nuclear near regions devoid of insulin (Fig. 5, A-C).
Regarding the C2 class, we next assessed the distribution of the Lamp2. Consistent with cathepsin localization, Lamp2 was observed in both areas close to the membrane containing insulin and in the perinuclear region without the hormone (Fig. 6A).
Because the C2 class contains numerous vesicle-associated proteins including Rabs and VAMPs, we focused on Rab37 and VAMP8. Double labeling experiments showed that VAMP8 mainly colocalizes to ISGs (Fig. 6B); the protein was also concentrated in perinuclear organelles devoid of insulin. Given that Rab37 antibody was not appropriate for immunofluorescence studies, we transiently overexpressed EGFPtagged Rab37 in INS-1E cells. Fig. 6C shows that EGFP-Rab37 was predominantly associated with ISGs.

Isolation of Insulin Secretory Granules
Subcellular fractionation has been widely used for proteomics analyses of different intracellular organelles and complexes such as mitochondria (32), Golgi network (33), lysosome (34), phagosome (35), endosome (36), peroxisome (37), or nucleoli (38). The main inconvenience of this approach is the potential presence of other subcellular structures that could share similar physical properties and comigrate in density gradients. Therefore, a complete purification of an organelle by subcellular fractionation is hardly possible. However, this approach is still the most efficient strategy to obtain enriched organelles of interest for further proteome studies (39). Regarding ISG purification, we first tested a continuous sucrose density gradient (0.45-2 M) that showed a poor separation of ISGs from the endoplasmic reticulum. Therefore, a two-step fractionation strategy with an initial gradient of Nycodenz followed by a Percoll solution was applied. This method generated a highly enriched fraction of ISG proteins as demonstrated by Western blot and electron microscopy experiments.

Proteomics of Insulin Secretory Granules
To date about 30 proteins have been reported as being associated with insulin granules. However, Guest et al. (40) have shown that a few hundred polypeptides could be detected by 2-DE gel analyses of an enriched ISG fraction. The main disadvantage of using 2-DE gels is the inability to analyze membrane proteins, which could be of primary importance in vesicles such as ISGs. In contrast, 1-DE gels coupled to liquid chromatography permitted a good extraction and separation of both soluble and insoluble proteins (24) and was therefore used in the present study. MS analyses were performed on the tryptic digests coming from 28 consecutive gel slices. This strategy identified 130 different proteins. Twenty of them have been reported previously to colocalize with ISGs, whereas further validation is required for the remaining 110.

The Structural Classification of ISG Proteins
According to the literature we could group the identified proteins in four structural classes: C1, intravesicular proteins (n ϭ 57); C2, membrane proteins (n ϭ 59); C3, novel proteins (n ϭ 4); and C4, other proteins (n ϭ 10). These four classes are discussed below.
Intravesicular Proteins-A large fraction of the 57 proteins is represented by secreted proteins (n ϭ 18), which include insulin, chromogranin A, betagranin, and secretogranin. A substantial number of proteins are represented by hydrolases (n ϭ 33) including different forms of cathepsin (B, D, and L) or glucuronidase. By immunofluorescence experiments, we located cathepsin B, D, and L in ISGs and in other vesicles most likely corresponding to lysosome. Several authors (18,41) have previously described the presence of hydrolases within ISGs. Kuliawat et al. (42) showed by electron microscopy that both cathepsins B and L are present in immature ␤-granules, whereas only the immunolabeling for cathepsin L (but not B) persists in mature granules. The authors could demonstrate that the sorting of these enzymes from the immature granules follows the same mechanisms as that in lysosomes, i.e. involving the mannose 6-phosphate receptor. These results might be consistent with the "sorting by retention" hypothesis in primary ␤-cells (43). In this hypothesis the immature secretory granules are the sorting point for proteins targeted either to mature granules or to lysosomes. On the contrary, in the "sorting for entry" hypothesis, the trans-Golgi network is the unique sorting point for ISG proteins and for proteins of other organelles (44). Alternatively to the above hypothesis, lysosomal proteins could come from the crinophagy process. Effectively the insulin content of ␤-cells is balanced through its biosynthesis, secretion, and degradation. Intracellular degradation of insulin is performed by crinophagy through the fusion of secretory granules with lysosomes (45). The three  alternative hypotheses are not exclusive because it is possible that the sorting by retention hypothesis only occurs in cells having regulated secretion such as ␤-cells (43). All together our results strongly support the sorting by retention hypothesis. Further enrichment of immature secretory granules will be required to investigate the role of well selected hydrolases in the early biogenesis of ISGs.
Membrane Proteins-A second large class of identified proteins is represented by membrane or membrane-associated proteins (n ϭ 59). We identified 14 different Rab proteins. Rabs are monomeric GTPases of the Ras superfamily that regulate different steps of vesicular trafficking. Although the role of Rab proteins in vesicle fusion is fairly well recognized, the precise task of such Rabs in transport is still unknown (46). Among these proteins only the Rab3 isoforms (26) and Rab27a (47) were shown to be associated with ISGs. Both proteins are involved in the regulation of insulin exocytosis together with their specific effectors such as Noc2, granuphilin, or the Rab3-interacting molecule RIM (48 -51). Among the 14 Rabs, we identified Rab12, which is associated with secretory granules of atrial myocytes and Sertoli cells (52,53), as well as Rab37, which is found on secretory granules in mast cells (54). Our immunofluorescence experiments revealed that overexpressed EGFP-Rab37 is associated with ISGs in INS-1E cells suggesting its functional role in the regulation of insulin secretion.
The VAMPs are members of the v-SNARE family, which participates in diverse intracellular docking/fusion events by pairing with their cognate t-SNAREs on the target membrane (55). We identified four VAMPs. VAMP2 and -3 are associated with ISGs (14), whereas VAMP7 and -8 are colocalized with late endosomes (56) and to zymogen granules of pancreatic acinar cells (57), respectively. Our confocal microscopy analysis demonstrated that VAMP8 is located on ISGs and other organelles probably corresponding to early endosomes as reported for the insulinoma MIN6 (57). We found other membrane proteins involved in the regulated exocytosis such as synaptotagmin 5 or annexin A4 as well as different forms of the vacuolar ATPase or voltage-dependent anion channel.
The finding of known lysosomal proteins in our preparation was not limited to hydrolases because Lamp2, a lysosomeassociated membrane protein, was identified as well. Using immunofluorescence, Lamp2 was detected on ISGs and other vesicular structures corresponding most likely to lysosomes.
Novel Proteins-For four of the 130 identified proteins we could not assign any biological function using sequence similarities, domain composition, or pathway information.
Other Proteins-Finally the function of a relative small proportion (n ϭ 10) of identified proteins like the fructosebisphosphate aldolase A or the peptidyl-prolyl cis-trans isomerase A has never been described in ␤-cells. Therefore, the validation and the association of these proteins with ISGs should be further investigated.
In conclusion, the present proteomics analysis of ISGs revealed 130 proteins. Confocal microscopy analysis of some selected proteins demonstrated their presence on ISGs. The validation of hydrolases and lysosomal proteins within ISGs supports the sorting by retention hypothesis. The association of Rab37 and VAMP8 proteins with ISGs was also highlighted for the first time suggesting an even more complex mecha- nism for vesicle trafficking than described previously in ␤-cells. In summary, these results clearly demonstrate that our strategy has determined a new subset of insulin secretory granule proteins that will further help to unravel the complex molecular anatomy and functional structure of insulin secretory granules.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Conserved α-Helix Acts as Autoinhibitory Sequence in AMP-activated Protein Kinase α Subunits*

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic α subunit and two regulatory subunits, β and γ. It had been reported that the mammalian AMPK α subunit contained an autoinhibitory domain (α1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the α subunit (α1-(313-335)), which includes a predicted α-helix, is responsible for α subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for α1 autoinhibition. An autoinhibitory structural model of human AMPK α1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 Å and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK α1-(1-394) with deletion of residues 313-335 (Δα394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Δα394, V298G, or L328Q mutants, which indicated that these AMPK α1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK α autoinhibitory function.

The mammalian AMPK ␣ catalytic subunit contains an N-terminal kinase domain (residues 1-312) and a C-terminal domain (residues 313-548), which includes an autoinhibitory domain (AID) and a binding domain responsible for binding the ␣/␥ subunits (34). Previous studies have identified residues 313-392 of the ␣1 subunit as the AID sequence and residues 313-473 of the ␣1 subunit as the ␣1 binding sequence (34,35). Alignment analysis of the AID sequence indicates that this segment is highly conserved in the AMPK subfamily of kinases. However, there is little conservation between AMPK and AMPK-related protein kinases, except for several conserved residues located in the ubiquitin-associated (UBA) domain, which is located closely following the kinase domain in many AMPK-related protein kinases and required for the activation and localization of these kinases (36). The mammalian AMPK ␣ catalytic subunits do not possess this UBA domain within the reported sequence at 313-392; instead, it plays an important role for ␣ subunit autoinhibition. Although the AID is known to be responsible for ␣ subunit inhibition, a more detailed autoinhibitory sequence and molecular basis for the autoinhibitory function of AMPK are unclear. Therefore, it was of interest to us to investigate which part of that domain (␣1, residues 313-392) in the mammalian AMPK ␣ catalytic subunit acts as the autoinhibitory sequence and determine how it exerts its autoinhibitory role. In this study, we identify that a conserved 23-residue sequence of the ␣ subunit (␣1, residues 313-335), including a predicted ␣-helix, acts as the autoinhibitory sequence in the AMPK ␣ subunit by means of truncation, deletion, and point mutation of the ␣1 subunit. Hydrophobic interaction between Val-298 and Leu-328, using our hypothesized autoinhibitory structural model of ␣1- , was confirmed by mutation and kinase assay. The hydrophobic and charged residues in the conserved ␣-helix might play important roles for the autoinhibitory activity of the ␣ subunit partly through tight packing of the ␣ autoinhibitory domain and maintenance of the conformation interacting with the kinase domain.

Plasmid Construction
The construction of all recombinant plasmids used in this study is detailed in Table 1. Cloning was performed using standard molecular biology techniques. The coding sequences of all human AMPK ␣1 or ␣2 subunit truncations, deletions, and mutants were amplified from cDNA of the human AMPK ␣1 or ␣2 subunit using relevant primers listed in Table 1 and cloned into pGEMEX-1 or pET28b vector. For the trimer ␣1/␤1/␥1 expression, the pETDuet-␤1/␣1 and pET28b-␥1 were constructed to co-transform into competent cells. The full-length genes of human AMPK ␣1 and ␥1 were amplified and then subcloned into pET28b vectors to construct pET28b-␣1 and pET28b-␥1 for expression of recombinant proteins with C-terminal polyhistidine tags, respectively. The human full-length AMPK ␤1 gene was cloned into the vector pETDuet-1 to form pETDuet-␤1, and then the AMPK ␣1 subunit gene and its upstream T7 promoter were subcloned from pET28b-␣1 and ligated into pETDuet-␤1 to form pETDuet-␤1/␣1.
For transfection plasmids construction using plasmid pcDNA3.1, the truncations were amplified from human AMPK ␣1 cDNA by PCR using relevant primers containing a consensus Kozak sequence for proper translation. To facilitate detection of the truncation expression in cells, a C-terminal His tag and Myc tag were incorporated into the truncations. All recombinant plasmids containing wild-type truncations, deletions, and mutations were verified by DNA sequencing.

Recombinant Protein Expression and Purification
E. coli cells containing the expression plasmids containing truncations, deletions, and mutant plasmids were cultured at 37°C until the absorbance of 600 nm reached 0.6, and then 0.1 mM isopropyl-1-thio-␤-D-galactopyranoside was added to induce protein expression at 22°C overnight. The cells were spun down and sonicated in lysis buffer (TL for nonfusion proteins: 50 mM Tris, pH 7.5, 1 mM DTT, and 1% Triton X-100). The nonfusion truncated proteins, deletions, and mutants were purified by loading the lysate supernatant onto a 20-ml HiPrep Q-Sepharose column (preequilibrated with 50 mM Tris, pH 7.5, 1 mM DTT) and eluted with a linear NaCl gradient (0 -1 M) in 50 mM Tris, pH 7.5, 1 mM DTT. All purified proteins were stored at Ϫ70°C.

Measurement of Enzyme Activities
In order to determine the phosphorylation time of kinase for an activity assay and due to inactive ␣1-(1-394), we chose ␣1-(1-312) as the active form incubated in a phosphorylation buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM DTT, 5 mM MgCl 2 , 200 M ATP (1.0 Ci of [␥-33 P]ATP/reaction) with CaMKK␤ (200 nM) at 30°C for the indicated times, at which aliquots were removed for determination of phosphate incorporation. ␣1-(1-312) was fully phosphorylated by incubation with upstream kinase for 4 h (Fig. 4A). In the following experiments for the kinase activity assay, all truncations, deletions, or mutations were preincubated in the phosphorylation buffer for 4 h before the enzyme assay. After recombinant proteins were fully phosphorylated by incubation with CaMKK␤ (200 nM) at The reaction mixture was then transferred to P30 filter paper and washed three times with 0.1% H 3 PO 4 . Radioactivity that had been incorporated in the proteins was determined by liquid scintillation counting in a Wallac Microbeta plate counter. Background radioactivity estimated from reactions conducted without enzymes was subtracted from sample radioactivities. All reactions were repeated in three independent experiments.

Cell Culture, Transfection, and Activity Analysis of Expressed AMPK in COS7
COS7 cells were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in 5% CO 2 . Transfection with plasmids was performed using Lipofectamine 2000 transfection reagent in cells that were

Plasmid
Protein expressed Primer sequence

␣-Helix Responsible for AMPK Autoinhibition
about 80% confluent. Briefly, COS7 cells in a 6-well plate were transiently transfected with 8 g of each DNA construct in Lipofectamine 2000 transfection reagent (0.5 l/g of DNA), using a GFP vector of pEGFP-C1 to monitor transfection efficiency when required and still cultured for about 24 h, allowing for AMPK expression. Before Western blotting analysis, the medium was aspirated, and the cells were rinsed with PBS and then lysed directly by 1ϫ SDS-PAGE loading buffer.

Glucose Uptake Assay in Human Hepatoma HepG2 Cells
HepG2 cells were cultured in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 5.5 mM D-glucose in 5% CO 2 at 37°C. For experiments, HepG2 cells were split into a 24-well plate, and the next day the cells, when grown to 70% confluence, were transiently transfected with 2 g of each plasmid/well in Lipofectamine 2000 transfection reagent (0.5 l/g DNA). A GFP vector of pEGFP-C1 was used as a control to monitor transfection efficiency. After transfection for 24 h, cells were washed once with PBS and then were incubated in either glucose-containing or glucose-free DMEM for 24 h. To determine the glucose uptake, the medium was switched to glucose-free HEPES-buffered saline (HBS buffer: 136 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO 4 , 1.2 mM CaCl 2 , 0.2% bovine serum albumin, 20 mM HEPES, pH 7.4). Glucose uptake was measured by adding a 50-l glucose mixture (1 mM 2-deoxyglucose and 0.5 Ci of 2-[ 3 H]deoxy-Dglucose in HBS) to 450 l of HBS followed by incubation for 3 min at 37°C. Nonspecific glucose uptake was measured by parallel incubations in the presence of 10 M cytochalasin B, which blocks transporter-mediated glucose uptake, and was subtracted from the total uptake in each assay. Uptake was terminated by quickly washing the cells three times with ice-cold PBS, and the cells were mixed with 200 l of cell lysis solution (0.1% Triton X-100 in phosphate-buffered saline). A portion of the cell lysate was used in a radioactivity assay performed using a liquid scintillation counter.

Western Blotting and Other Analytical Procedures
The above prepared samples were subjected to 8 -12% SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The quantities of the protein and phosphorylation were detected by the following primary antibodies: anti-AMPK-␣ antibody and anti-phospho-AMPK␣-Thr-172 antibody (1:1,000 dilution), anti-phospho-ACC-Ser-79 antibody (1:1,000 dilution), anti-ACC antibody (1:1,000 dilution), anti-Myc antibody (1:2,000 dilution), and anti-␤-actin antibody (1:5,000 dilution). The total protein of each sample was quantified by ␤-actin. The signal was detected by a chemiluminescence kit and analyzed by densitometric evaluation using an imaging system and analyzing software (Shanghai Furi Science & Technology Co.). Protein concentration was measured using the Bradford method.

Modeling of AMPK ␣1 Autoinhibitory Structure
For Homology Modeling-The crystal structure of yeast AMP-activated protein kinase homologous Snf1p (Protein Data Bank code 2EUE) was used to construct the kinase domain (residues 1-280) of human AMPK ␣1 by utilizing the program MODELLER. The sequence identity between these two proteins is 56%, and the positive score is 71%, whereas for the autoinhibitory domain (residue 290 -335), the sequence searching against the Protein Data Bank did not result in any informative alignment. Therefore, the secondary structure prediction was conducted within the server (available on the World Wide Web at www.compbio.dundee.ac.uk/ϳwww-jpred/) performed by the built in program Jpred, which suggested that this domain contained three ␣-helices. Then functional search found that the ubiquitin-associated domains of many kinases contain such structural features. Also, as provided by Jaleel et al. (36), the sequence alignments between AMPK␣1 and microtubule affinity-regulating kinases (MARKs) were adopted to build this autoinhibitory domain. Due to the low sequence identity, after the comparative modeling by MODELLER, the CHARMM program was used to solvate this domain in a water box extending 10 Å from any atoms of the domain. Then the energy minimization was performed in about 1000 steps with the conjugate gradient method to relax the autoinhibitory domain. Following this, 10-ps molecular dynamics was carried out to further explore the conformation of the domain by constraint of the backbone atoms with a force constant of 10 Kcal/mol Å 2 .
For Docking-As suggested in the crystal structure of protein kinase MARK/Par-1, the ubiquitin-associated domain in MARK (equivalent to the autoinhibitory domain in AMPK) is located at the back side of the N-terminal lobe of the kinase domain. Thus, the complex of autoinhibitory domain and kinase domain of AMPK was formed according to the coordinates of the crystal structure of MARK (Protein Data Bank code 1Y8G). To release the crash between these two domains, the CHARMM program was used again to minimize the complex with the procedure based on the program RDOCK, first by only minimizing the van der Waals interaction and keeping each domain as a rigid body and then by adding the electrostatic force to minimize the complex while the distances of each domain were still fixed; after that, the side chains of the two domains were allowed to move under the minimization of the bonded and nonbonded interactions as normally conducted in the CHARMM force field. Finally, the complex was used to analyze the interaction between the two domains and explain the experiment results at the atomic basis.

Statistical Analysis
The results were expressed as the means Ϯ S.E. Significance was analyzed using a two-tailed unpaired Student's t test. p Ͻ 0.05 was considered significant.

RESULTS
Analysis of Truncated ␣1 Subunit of Human AMPK-Alignment of all available DNA sequences of the AMPK subfamily within the residue sequence ␣1-(271-394) of human AMPK, which succeeds the kinase catalytic domain, indicated that this segment was highly conserved. There were 62% identity and 77% positives between human AMPK ␣1 and ␣2 isoforms, with 100% identity between human AMPK ␣1 and rat AMPK ␣1 and 16% identity between human AMPK ␣1 and the yeast Snf1p kinase homolog (Fig. 1A). There are several conserved segments among members of the AMPK subfamily within this ␣-Helix Responsible for AMPK Autoinhibition region. Due to the conserved autoinhibition in AMPK subfamily kinases, the detailed mechanism must rely on conserved residues or dimensional structure. Sequence alignments suggest that these segments might play an important role for the autoinhibitory function of the AMPK ␣ catalytic subunit. Although the catalytic domain between AMPK and the AMPK-related protein kinases is much conserved, there is only weak conservation within the residue sequence equivalent to ␣1-(271-394) of human AMPK.
In previous studies, Crute et al. (34) identified residues 313-392 in the mammalian AMPK ␣1 catalytic subunit as an autoinhibitory sequence and ␣1(1-312) as constitutively active, whereas ␣1(1-392) and full-length ␣1 catalytic subunit have little activity. A more detailed autoinhibitory sequence and mechanism remains unclear. In order to define the location of the autoinhibitory domain more precisely, we constructed a series of truncations of human AMPK ␣1-(1-394) under the strategy to gradually remove the conserved sequence and assessed their activity. Seven ␣1 C-terminal deletion constructs were created in pGEMEX-1 plasmid in order to truncate the human AMPK ␣1 subunit at residues 394, 377, 367, 351, 341, 335, or 312 (Fig. 2). The truncations were purified to near homogeneity by Q-Sepharose anion exchange chromatography (Fig. 3). All truncated proteins were fully phosphorylated at Thr-172 after incubation with CaMKK␤ to a similar extent, and then the kinase activity was assessed by incorporation of [ 33 P]phosphate into the SAMS peptide under a typical assay described under "Experimental Procedures." As depicted in Fig. 4, the isolated kinase  ␣-Helix Responsible for AMPK Autoinhibition domain (residues 1-312) of AMPK ␣1 catalytic subunit was constitutively active when expressed in E. coli cells, which was consistent with previous studies (19,34,40), whereas proteins truncated to residues 394, 377, 367, 351, 341, and 335, exhibited little activity compared with the high activity of ␣1-(1-312) truncation. As a result, the ␣1-(1-335) fragment was defined as the shortest truncation that retained the autoinhibitory activity, indicating that residues 313-335 of the human AMPK ␣1 subunit might act as a critical sequence responsible for autoinhibition.
Due to the unsuccessful attempts to obtain nondegraded fulllength ␣1 subunit from bacteria cells, we could not examine the activity of the full-length ␣1 subunit. It was reported previously that the full-length ␣1 monomer, in the absence of the ␤/␥ regulatory subunits, obtained from transiently expressed mammalian cells, showed little kinase activity (13,34). In this study, deletion of residues 313-335 in the full-length human AMPK ␣1 subunit did increase activity, ϳ2-fold higher than ␣1-(1-394) but only one-third lower than that of ␣1-(1-394, ⌬313-335) (Fig. 5).
Effect of Mutation of Residues 320 -335 on ␣1-(1-394) Autoinhibitory Activity-According to the enzyme's secondary structure prediction using the Jpred software (available on the World Wide Web at www.compbio.dundee.ac.uk/ϳwwwjpred/), the 23 residues of ␣1-(313-335) might contain a conserved ␣-helix structural fold between residues 320 and 335. The presence of more conserved charged residues and hydrophobic residues in this predicted ␣-helix raised the possibility that these residues might be responsible for the enzyme's autoinhibitory activity. In order to test which residues were critical, we next assessed how mutation of each of the residues affected the catalytic activity of ␣1-(1-394). Each mutant of ␣1-(1-394) was purified to near homogeneity (data not shown). Several mutations were chosen to examine their phosphorylation level by immunoblotting with phospho-AMPK␣-Thr-172 antibody, and they were phosphorylated by CaMKK␤ to a similar extent after incubation with phosphorylation buffer for 4 h (Fig. 6A). It was found that these mutants, except for A323R, H327E, and I335N, had higher kinase activities than wild-type ␣1-(1-394) by measuring the kinase activity toward AMPK peptide substrate SAMS (Fig. 6B). Interestingly, mutation of the conserved Asp-320, Leu-328, Arg-333, or Arg-334, especially the hydrophobic residue Leu-328, markedly enhanced the catalytic activity of the enzyme, indicating that the charged and hydrophobic residues in 320 -335 of the ␣1 subunit might play important roles for the autoinhibition.

Construction of a Structural Model for Human AMPK ␣1 Subunit, Including the Kinase Domain and Autoinhibitory
Domain-Recently, the homologous yeast Snf1 kinase domain was crystallized, and the structure was determined by x-ray crystallography. It provided an opportunity to model the kinase domain of human AMPK ␣1 as described under "Experimental Procedures." The secondary structure prediction of residues 280 -335 suggested that this segment has several ␣-helix structures. Then a structural homology search was conducted using the model of the AMPK kinase domain (residues 1-280) against the AMPK-related kinases in the Protein Data Bank. It was found that the structure of human MARK2 (Protein Data Bank code 1Y8G) contained a similar scaffold to the kinase catalytic domain and also contained a three-␣-helix UBA domain. This is consistent with other studies, such as the UBA domain of HHR23A. Then the structure of residues 290 -335 of AMPK ␣1 was constructed based on the MARK2 UBA domain and presented in Fig. 7. We modeled two structures rather than one partly due to the sequence conservation and the lack of the ␣1-(280 -290) homologous region in MARK2. To form a com-

␣-Helix Responsible for AMPK Autoinhibition
plex with the kinase domain and autoinhibitory domain of AMPK ␣1-(1-335), the two built models were adapted and superimposed on the structure of MARK2. Similar to the MARK2 protein, the autoinhibitory sequence (residues 290 -335) was formed by three ␣-helices and located on the backside of the N-terminal lobe of the kinase catalytic domain. Due to the impact of residue Leu-328 of the human AMPK ␣1 catalytic subunit on the autoinhibitory function and its conservation in most of AMPK-related kinases (36), its location was assessed by our complex model of AMPK ␣1. As Fig. 8 shows, Leu-328 might interact with Val-298, which is located in the noncatalytic domain according to their distance of around 4.3 Å between the first helix and the second helix of the AID. In order to determine whether the Val-298 amino acid interacts with Leu-328 to play a similar role for autoinhibition, Val-298 was mutated to glycine in ␣1-(1-394). It was confirmed that V298G weakened the autoinhibition by enhancing the enzyme activity, similar to that of L328Q (Fig. 8). Another two mutants were also suggested by the complex model, being residues located on the interface of kinase domain and autoinhibitory domain. However, the point mutation results showed that these two mutants had only low levels of kinase activity similar to that of wild-type ␣1-(1-394), which indicates that Leu-39 and Tyr-82 are not involved in the interactions between the kinase domain and the AID (Fig. 8).

Effect of Overexpression of ⌬␣394, V298G, or L328Q on Glucose Uptake in HepG2
Cells-Previous studies with multiple cell lines have demonstrated that glucose deprivation activated AMPK (41)(42)(43)(44)(45). To investigate the effect of deletion AMPK ␣ mutant on liver cellular metabolism function, we assessed glucose deprivation-induced glucose uptake in HepG2 cells that overexpressed ⌬␣394, V298G, or L328Q mutant. Following glucose deprivation, 2-deoxyglucose uptake was measured as described under "Experimental Procedures," and the results are shown in Fig. 10. Glucose deprivation significantly enhanced 2-deoxyglucose uptake into HepG2 cells about 1-fold, compared with that in the control containing 5.5 mM glucose (p Ͻ 0.01). Although overexpression of ␣1-(1-394) in the cells, after glucose deprivation, did not affect 2-deoxyglucose uptake, overexpression of ⌬␣394, V298G, and L328Q significantly increased 2-deoxyglucose uptake by 25, 32, and 36%, respectively, as compared with that in the cells that overexpressed empty vector as control (p Ͻ 0.05), indicating that the deletion AMPK ␣ mutant (⌬␣394), V298G, and L328Q are also constitutively active to promote glucose uptake in hepatocytes after glucose deprivation.

DISCUSSION
Although the exact mechanism of autoinhibition in the AMPK ␣ subunit is unclear, it is possible that the autoinhibitory domain binds to the kinase domain in an inactive conformation, with the ␣ subunit interfering with kinase substrate binding and catalytic function. Similar autoregulation has been shown to exist in other protein kinases, such as the Ca 2ϩ / calmodulin-dependent protein kinases, whose C-terminal domain binds into the catalytic cleft (46, 47), cAMP-dependent

␣-Helix Responsible for AMPK Autoinhibition
protein kinase, protein kinase C, mitogen-activated protein kinases (48 -50), or MARK isoforms, which have an insertion of a third helix of the UBA domain through interaction with the N-terminal lobe (51). The results obtained from the present studies using truncation, deletion, and mutagenesis methods provide important information about the mechanism of AMPK ␣ subunit autoinhibition.
An important observation made in this study is that, in the truncated form of the human AMPK ␣ subunit, ␣1-(1-335) is sufficient to maintain an inactive, autoinhibited state, suggesting that residues 313-335 play an autoinhibitory role. This result extends the findings of previous truncation studies that had indicated a role for residues 313-392 in autoinhibition of the mammalian AMPK ␣ catalytic subunit (19,34). The ␣1-(313-335) residue sequence is highly conserved in the AMPK subfamily (Fig.  1). The removal of this segment results in human AMPK ␣1-(1-394) or ␣2-(1-398) being constitutively active (Fig. 5). However, deletion of residues 313-335 in the full-length ␣1 subunit did not fully activate the kinase domain. We speculate that the extreme C-terminal sequence 394 -550 of the ␣1 subunit might also interact with the kinase domain, resulting in a partially active conformation of ␣1-(1-550, ⌬313-335), but this detailed model was not constructed. Surprisingly, the ␣1 subunit without residues 313-335 was obtained without degradation when expressed in bacterial cells (Fig. 3). Previous studies have shown that the full-length ␣1 monomer of mammalian AMPK, when expressed in mammalian cells, had a more rapid turnover than the ␣1 fragment truncated to 312 or to 392, whereas binding of the ␤/␥ regulatory subunits to the C-terminal 313-548 sequence of ␣1 catalytic subunit stabilizes the ␣1 subunit, resulting in increased expression and decreased turnover (34). It is possible that residues 313-548 may be targeted by cellular proteins, such as ubiquitin, for rapid degradation. We have also found this degradation of full-length ␣1 subunit when expressed in bacterial cells lacking the ubiquitinmediated pathway, so there may also exist an autodegradation mechanism within the cellular AMPK ␣ catalytic subunits. However, the degradation mechanism remains unclear and will be further investigated. These data demonstrate that residues 313-335 comprise the exact autoinhibitory sequence in the human AMPK ␣1 subunit and not only are responsible for the kinase autoinhibitory function but may also be responsible for full-length kinase degradation.
Secondary structure prediction indicates that this autoinhibitory sequence region forms an ␣-helix between residues 321 and 329. According to the structural model we constructed, the region linked to the catalytic domain assumes an extended conformation, and this region consists of three ␣-helices at the back side of the catalytic domain, with the third ␣-helix being inserted by interaction with the catalytic domain, similar to the UBA domain of human MARK2. Moreover, the third ␣-helix, as an autoinhibitory domain, may also play an important role for keeping the cohesion of this three-short helix conformation

␣-Helix Responsible for AMPK Autoinhibition
through interaction with the first ␣-helix. The removal of this ␣-helix as an autoinhibitory sequence might alter the open (inactive) conformation into an active conformation and significantly enhance the kinase catalytic ability. It is also speculated by the structural model that this ␣-helix might be associated with the large lobe of the kinase domain to interfere with substrate binding. The ␣1-(313-473) sequence, including residues 313-335, is required for ␤1 binding (35). Once it is binding with the AMPK regulatory subunits ␤/␥ to detach the autoinhibitory domain from the kinase domain, the full-length ␣ catalytic subunit would exhibit the maximal kinase activity. Therefore, the autoinhibitory conformation of the full-length ␣ subunit induced by the ␣1 (313-335) sequence will be disrupted through interaction between the AMPK regulatory subunits ␤/␥ and the ␣1-(313-335) sequence. These results provide insight into the interrelationship between the autoinhibition of the AMPK ␣ subunit and the activation of the AMPK ␣ subunit by AMPK regulatory subunits.
The hydrophobic and charged residues in the conserved ␣-helix play important roles for the autoinhibitory activity of the ␣ subunit, as indicated by the point mutation experiment. Many constitutively active mutants underscore that this conserved ␣-helix is the determinant of the autoinhibition (Fig. 6). Most of the mutations resulted in the generation of a modest level of the kinase activity, but mutation of the charged residues to alanine resulted in a significant increase in the kinase activity. It is possible that these charged residues are involved in mediating charge-based interactions between the autoinhibitory domain and the kinase domain and serve to anchor the ␣-helix in its correct position. However, this interaction was not a pseudosubstrate interaction like cAMPdependent protein kinase, protein kinase C, or Ca 2ϩ /calmodulindependent protein kinase regulation (46,(52)(53)(54), because the isolated sequence ␣1-(313-335) did not inhibit the kinase domain activity (data not shown). Among all point mutations, the maximum activity of the L328Q mutant reaches about three-quarters the activity of ␣1-(1-394, ⌬313-335), suggesting that the Leu-328 residue is the key residue responsible for autoinhibition of the ␣1 subunit activity. However, it is not responsible for full-length ␣1 subunit degradation due to the marked degradation of L328Q-␣1-(1-550) when expressed in bacterial cells (data not shown). From our speculative model constructed by homology modeling, it was demonstrated that Leu-328 has a hydrophobic interaction with Val-298 within the link of the kinase catalytic domain and autoinhibitory domain (Figs. 7 and 8). Through the kinase assay of the V298G mutant of AMPK ␣1 subunit, it strongly weakened the autoinhibition by enhancing the enzyme activity similar to that of L328Q. This reinforced that Leu-328 forms hydrophobic intra-AID interactions directly with Val-298 to further stabilize the AID internal cohesion, which retains it in the autoinhibitory conformation to interact with the kinase domain, as shown in our autoinhibitory structural model (Fig.  7). Based on the alignment of the autoinhibitory sequence (residues 313-335) of the human AMPK ␣1 subunit with that of AMPK-related kinases (Fig. 1), it was found the Leu-328 is one

␣-Helix Responsible for AMPK Autoinhibition
of the most conserved residues across the whole superfamily, as was Val-298. Similar to the UBA domain of human MARK2, three conserved leucines near the end of helix ␣3 of UBA also play critical roles in MARK2 stability, whereas the functions of the AID in AMPK catalytic subunits are absolutely distinct from the UBA domain of AMPK-related kinases, which is required for the activation of kinases. The kinase activity assay of the mutants of L328Q and V298G shed light on the autoinhibitory mechanism and encourage us to use the complex model to assess the interacting residues in the kinase catalytic domains. Because the L328Q or V298G mutants could not fully abolish the kinase inhibition to the same extent as ⌬␣394, it is suggested that these charged residues, such as Asp-320, Arg-333, Arg-334, or others in this ␣-helix, may make hydrogen bond contacts with the kinase domain, which could be concluded from the activity of the mutants in contrast to wild-type. From the superimposition of the AMPK ␣1 model on the MARK2 structure, it is clearly demonstrated that the residues Leu-39 and Tyr-82 are located on the interface between the kinase catalytic domain and AID and would interact strongly with the AID. Our attempts to demonstrate the anticipated interaction residues by mutation of these residues (L39G and Y82G) in the kinase domain, however, were frustrated by the kinase inhibition activities from these mutants (Fig. 8). One possibility is that these mutation sites in the kinase domain might not interact with the autoinhibitory domain. As studied by Jaleel et al. (36), small angle x-ray scattering experiments showed that the UBA domain of MARK2 was located close to the substrate binding site to inactivate the kinase. This may indicate that the conformation the UBA domain adopted is different in solution than in crystal lattice. Other possibilities may also exist; e.g. the mutation on these residues may destroy the integrity of the kinase domain, so the mutants could not fold properly and therefore were functionally inept. Recently, Nayak et al. (37) have reported a crystal structure of the kinase domain of inactive yeast Snf1 presented as a homodimer in solution and in yeast cells whose interface involved the kinase domain segment through hydrophobic interactions resulting in the inhibition of substrate binding. The high conservation of most of the dimer interface residues within the Snf1/AMPK and AMPKrelated kinase family indicates that the kinase domain of AMPK or AMPK-related kinases would also form into dimers. However, the conserved ␣-helix in the AID of AMPK ␣ subunit, located at the back side of the cleftlike UBA domain of human MARK2, may not be involved in the dimer interaction. These constitutively active deletions or mutations might not disrupt the ␣ subunit dimer conformation.
Further cellular experiments provide confirmation that the constitutively active deletion AMPK ␣ mutant (⌬␣394), V298G, and L328Q function as in vitro. When transfected into COS7 cells, ⌬␣394, V298G, and L328Q maintain constitutive activity, resulting in an increase in the phosphorylation level of the AMPK downstream substrate, ACC, compared with control, respectively (Fig. 9). When transfected into HepG2 cells, following glucose deprivation, which normally activated AMPK, ⌬␣394, V298G, and L328Q significantly enhanced cellular glucose uptake, compared with control, respectively (Fig.  10). The above cellular results confirmed that deletion of this conserved ␣-helix is sufficient to keep the AMPK ␣ catalytic subunit active, in which the conserved Leu-328 residue has strong interaction with the residue Val-298, which is outside of that autoinhibitory domain. As our speculative modeling suggested in Fig. 7, the strong hydrophobic Val-298/Leu-328 interaction may stabilize the conformation of the three ␣-helices in the autoinhibitory domain and may aid the interaction between the N-terminal kinase domain and the autoinhibitory domain.
In summary, we have for the first time elucidated the exact autoinhibitory sequence, a conserved ␣-helix in the AMPK ␣ catalytic subunit isoforms, and our results support the autoinhibitory model we constructed, in which the autoinhibitory domain folds into an autoinhibitory conformation predominantly by hydrophobic interactions between Leu-328 and Val-298 residues or others. This special conformation will open a new regulatory mechanism for kinase autoinhibition. Furthermore, three-dimensional structural studies will hopefully reveal additional details about the AMPK autoinhibition mechanism and apply this mechanism to the autoregulation of Snf1p kinase and the regulation by the ␤/␥ subunit.