Pharmaceutical Relevant Proteins; Studies of RBP4 and Kvβ2. Gavin Mooney School of Food Science and Environmental Health 2011 Thesis Submitted in Partial Fulfilment of Examination Requirements Leading to the Award B.Sc. Pharmaceutical Technology Dublin Institute of Technology Supervisor: Dr. Barry Ryan Asst. Supervisor: Ms. Alka Singh, M.Sc.
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Pharmaceutical Relevant Proteins; Studies of
RBP4 and Kvβ2.
Gavin Mooney
School of Food Science and Environmental Health
2011
Thesis Submitted in Partial Fulfilment of Examination Requirements Leading to the
Award
B.Sc. Pharmaceutical Technology
Dublin Institute of Technology
Supervisor: Dr. Barry Ryan Asst. Supervisor: Ms. Alka Singh, M.Sc.
ii
Declaration I Certify that this thesis which I now submit for examination for the award B.Sc.
Pharmaceutical Technology is entirely my own work and has not been taken from
the work of others, save and to the extent that such work has been cited and
acknowledged within the text of my work.
This thesis was prepared according to the regulations provided by the School of
Food Science and Environmental Health, Dublin Institute of Technology and has
not been submitted in whole or in part for another award in any Institute or
University.
The Institute has permission to keep, lend or copy this thesis in whole or in part,
on condition that any such use of material of this thesis is duly acknowledged.
3.5 Bradford Method Standard Curve....................................................................47
x
List of Figures:
Fig.1.1 3-D Protein representation of the RBP4 structure.
Fig.1.2 Schematic of aldehyde-dismutation in Kvβ2
Fig.1.3 Structural features of Kvβ
Fig.1.4 Chemical structure of Rutin
Fig.1.5 Chemical structure of Resveratrol
Fig.1.6 Chemical structure of Quercitin
Fig.2.1 Gradient elution method employed for the HPLC Analysis of
Resveratrol and Rutin
Fig.3.1 PCR UV Photograph of PCR #1
Fig.3.2 PCR UV Photograph of PCR #2
Fig.3.3 PCR UV Photograph of PCR #3
Fig.3.4 PCR UV Photograph of PCR #4
Fig.3.5 PCR UV Photograph of PCR #5
Fig.3.6 Elution profile of purified Kvβ2
Fig.3.7 Chromatogram showing a control reaction for Rutin experiment
Fig.3.8 Chromatogram showing a control reaction for Rutin experiment
Fig.3.9 Concentration dependant inhibition of Kvβ2 by Rutin
Fig.3.10 Chromatogram showing a control reaction for Quercitin experiment
Fig.3.11 Chromatogram showing a control reaction for Quercitin experiment
Fig.3.12 Concentration dependant inhibition of Kvβ2 by Quercitin
Fig.3.13 Chromatogram showing a control reaction for Resveratrol experiment
Fig.3.14 Chromatogram showing a control reaction for Resveratrol experiment
Fig.3.15 Concentration dependant inhibition of Kvβ2 by Resveratrol
Fig.3.16 Concentration dependant percentage inhibition of Kvβ2 by Rutin
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Fig.3.17 Concentration dependant percentage inhibition of Kvβ2 by Quercitin
Fig.3.18 Concentration dependant percentage inhibition of Kvβ2 by Resveratrol
Fig.3.19 Flourometric data showing the binding of Rutin to Kvβ2
Fig.3.20 Flourometric data showing the binding of Quercitin to Kvβ2
Fig.3.21 Flourometric data showing the binding of Resveratrol to Kvβ2
Fig.3.22 BSA standard curve
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List of Tables:
Table 1. Description of Plasmids
Table 2. Details of Primers Used
Table 3. Comparison of Agarose Volumes Used
Table 4. Details of Primer concentration and cDNA volumes used in PCR 1
Table 5. Temperature gradient and tube layout for PCR 2.
Table 6. Temperature gradient and tube layout for PCR 5.1. (Machine 1)
Table 7. Temperature gradient and tube layout for PCR 5.2. (Machine 2)
Table 8. Outline of reaction mixtures for Bradford Method
1
Chapter 1:
Introduction
2
1.0 Introduction The aim of this project is to study the hRBP4 protein through cloning,
expressing and purifying of the protein in order to perform a selection of tests on
the protein for stability and characteristics. As will be seen throughout this report,
the optimisation process for the PCR of hRBP4 was recurrently unsuccessful and
as a result, it was decided to focus on the standardization of an experiment being
carried out by a PhD student on a new protein, Kvβ2. This protein, a subunit of the
Kv1 protein, is involved in the regulation of the Shaker potassium channels of the
body. In this study, three compounds; Rutin, Quercitin and Resveratrol have been
identified as inhibitors of the kinetic mechanism for aldehyde dismutation that has
been proposed for Kvβ2. For this study, the Kvβ2 was expressed and purified for
HPLC analysis of the inhibitory studies.
1.1 Retinol Binding Protein 4
It has been reported that protein human Retinol Binding Protein-4 (hRBP4) is
linked with insulin resistance when serums levels are elevated (Graham, Yang,
Blüher, et al., 2006). As such, this protein has a casual function in Diabetes
Mellitus Type 2 (DM2). The RBP4 protein is secreted from hepatocytes and
adipocytes. Studies have been conducted on RBP4 regarding its role in the
reduced expression of the glucose transporter-4; GLUT4, an adipocyte which is
responsible for the post-digestive function of glucose uptake through the insulin-
mediated conscription of the GLUT4 transporter to the cell (Graham, et al., 2006).
RBP4, in serum, is now recognised as an adipokine linked with diminishing
hepatic and peripheral insulin sensitivity and therefore increased hepatic
gluconeogenesis (Craig, Chu and Elbein, 2006). It has been identified that the
RBP4 gene is located close to a region linked with type 2 diabetes (DM2) and as
such may be the reason for increased susceptibility to DM2 and the reduction in
insulin sensitivity (Craig, et al., 2006). An in vivo study by Craig, et al. (2006)
aimed at knocking out the adipose-specific GLUT4 in which mice acquired
muscle and hepatic insulin resistance and as such also showed an elevated serum
concentration of RBP4. Elevated RBP4 levels were noted in a population study of
subjects with impaired glucose tolerance and these serum levels dropped in
individuals with exercise-mediated improved insulin sensitivity, as well as the
3
serum levels showing an inverse relationship to insulin sensitivity in individuals
with a family history of DM2 (Graham, et al. 2006). The elevated serum
concentration of RBP4 was also reported by Craig et al. (2006) in individuals with
diabetes relative to euglycemic therapy. While insulin resistance is a vital
accomplice to DM, it can also provide a risk of cardiovascular disease (CVD) and
artherosclerosis indirectly via a pathophysiologic link and thus elevated serum
RBP4 is associated with CVD risk factors and metabolic syndrome; giving the
potential for RBP4 to be used as a predictor to DM2 (Suh, Kim, Cho, Choi, Han
and Geun, 2009). Reports have shown that RBP4 serum concentrations correlated
with diastolic blood pressure, fasting glucose levels and age; all factors associated
with DM (Suh, et al. 2009). Suh, et al. also reported the implications this may
have for lipid metabolism and insulin action. In this same study, it was reported
that serum RBP4 levels may correlate to age-induced insulin resistance (IR) as
well as independently being associated with fasting glucose levels. Women over
50 years of age consistently possessed higher serum RBP4 levels in the study by
Suh, et al; attributed to the reduced levels of Oestrogen during menopause which
leads to changes in the fat amounts in the body and visceral fat increases, thus
causing lipid metabolism changes and increasing RBP4 serum concentrations. The
link between RBP4 levels and fasting glucose concentrations can possibly be
explained through the mechanism which causes the induced expression of the
gluconeogenic enzyme
phosphoenolpyruvate carboxykinase by RBP4 via the liver which leads to RBP4-
induced IR in the liver (Suh, et al. 2009).
Fig.1.1: 3D representation of Retinol Binding Protein-4.
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Serum RBP4 is reported as being preferentially being expressed in visceral fat
as opposed to expression in subcutaneous fat (Klöting, Graham, Berndt, et al.
2007). RBP4’s correlation with Transthyretin, which stabilizes circulating RBP4
and prevents it from being removed from plasma by glomerular filtration, was
used as an indicator to show the increase in visceral RBP4 in obese subjects, with
DM2 and impaired glucose tolerance (IGT), and was reported to be 35% over the
normal serum concentrations and therefore linking visceral adiposity and visceral
fat to RBP4 concentrations in the serum (Klöting, et al. 2007). Studies have
shown that RBP4 can be a clinical biomarker of IR in patients who present with a
range of clinical presentations, however it has also been reported that no evidence
has been found to back up this correlation and as such Chen, Wu, Chang and Tsai,
et al reported in 2009 the correlation between renal function rather than DM2.
Their study concluded that there was an expected inverse correlation between
RBP4 and uric acid, excreted by the kidneys due to the proven link between RBP4
levels and estimated glomerular function (eGFR), hence the relationship between
RBP4 and renal function in patients with DM2 (Chen, et al 2009). This may be an
indirect biomarker of IR and DM2 for RBP4 levels.
However, using a large study cohort, Lewis, Shand, Elder and Scott reported
in 2008 that RBP4 in the plasma may not be a functional biomarker of IR. In their
study of 285 fasting patients, some of whom had diabetes and some with no
diabetes but with varying levels of IR, the data observed did not provide any
relationship between RBP4 levels and IR and even body mass index (BMI),
percentage body fat and waist circumference as RBP4 levels were not
significantly higher in individuals with DM compared to those without. Thus also
putting into question; the correlation between lipid metabolism and plasma RBP4
levels. As already stated glomerular dysfunction can increase the levels of
circulating RBP4 and it has been noted that RBP4 levels in DM2 patients have
been affected by early nephropathy (Lewis, et al. 2008). RBP4 in this instance
may still be used as a biochemical marker of IR and there is still no evidence on
the contrary that GLUT4 levels reduce with a positive inverse in RBP4 expression
and secretion into serum. As such this is an analytical approach that may be used
to identify IR through RBP4 plasma concentration.
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The standard treatment, according to the British National Formulary 2010, in
DM2 to reduce peripheral IR is the administration of Pioglitazone or
Rosiglitazone; both of which are Thiazolidinedione (TZD)-classed medications.
These drugs are typically prescribed in combination with Metformin or a
Sulphonylurea in order to reduce IR, as stated, by targeting the regulation of
adipocyte function from which RBP4 levels can be managed. TZD act on the
adipocyte function through adipocyte differentiation and adipocyte gene activation
as they are a synthetic peroxisome proliferator-activated receptor-gama (PPAR-γ)
ligands (Sasaki, Nishimura and Hoshino, et al 2007). TZDs are broadly used to
reduce hyperglycaemia and have been reported to increase serum adiponectin
significantly better than Sulphonylureas (Lin, et al. 2008). Due to the minimal
selectivity of the PPAR-γ modulator, these TZD drugs can provide a undesired
side effects such as gastrointestinal disruption, obesity and oedema and there for
more attention has been focused on food based compounds such as Anthocyanins
like Cyanidin 3-glucosides (C3G) for a more effective management of DM2 and
metabolic syndrome through their efficacy in the modulation of the GLUT4 –
RBP4 system as well as inflammatory adipocytokines which result in the
improvement of hyperglycaemia and insulin sensitivity of patients with DM2
(Sasaki, et al 2007). Anthocyanins are water-soluble, plant based chemicals as due
to their abundance in the plant kingdom it is suggested that high amounts of
Anthocyanins are ingested through plant-based diets, hence the ingestion of
C3G’s which Sasaki et al has reported to be a suppressor of RBP4 expression in
white adipose tissue with a reported 47% reduction in serum RBP4 of the study’s
group compared to the control group of diabetic mice. The treatment of these
diabetic mice with C3G also showed an increase in the expression of GLUT4
transporters, likely to be due to the reduced expression of RBP4, leading to better
insulin sensitivity. Dietary C3G treatment also proved to increase insulin
sensitivity but with no significant affect on the expression of adiponectin and its
receptors; leading to observations that polyphenols may inhibit α-glucosidase
activity although the amelioration of IR by C3G is not due to inhibition of α-
glucosidase activity (Sasaki et al 2007). This suggests a new class of drug and
dietary treatment for DM2 and metabolic syndrome in respect of the management
of IR.
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1.2 Kvβ2: The Subunit of Kv1 Potassium Channels
Kvβ2 is a cytosolic protein; a subunit of Kv1 potassium (K+) channels which
are belong to the voltage-dependant ‘Shaker family’ of potassium channels.
Potassium channels are known to be widely dispersed ion channels within the
body, particularly in the Central Nervous System (CNS) and these are present in a
large number of living species (Kelly, 2010). The high abundance of Kvβ in the
nervous system may attribute to channel regulation in the myocardial cell and
impact on the action potential of same. The channels also provide a vital function
of establishing resting membrane potential and regulation of frequency during
action potential (Hille, et al. 1991). Such electrical activity is vital for the
functioning of process in excitable cells such as neurons and muscle (Long, et al.
2005). The Kvα subunit, Kvβ associates with the cytoplasmic aspect of the Kvα
protein and these do not contribute to ion conductivity however they do regulate
channel activity; the Kv channels have been shown to be responsible for the
regulation of K+ flow through cell membranes upon changes in the potential of the
membrane (Weng, et al 2006). These channels form transmembrane pores which
can be found in a variety of cell types in which they regulate the electrical
function and signalling processes among other physiological processes (Di
Costanzo, et al. 2009).
Fig.1.2: A mechanism for aldehyde dismutation in Kvβ2 as proposed by Alka, et al. 2010. RCHO is an aldehyde, RCH(OH)2 is its corresponding hydrate, RCOOH and RCH2OH are the corresponding alcohol and carboxylic acid respectively. The dismutation of aldehyde substrate consists of two
coupled half reactions. In the first half (the upper pathway), hydrated aldehyde is oxidised irreversibly to the corresponding carboxylic acid forming ENADPH. In the second half reaction (lower pathway), another molecule free aldehyde binds to the ENADPH complex and is reduced reversibly to corresponding alcohol. Hence, aldehyde is dismutated into equimolar concentration of corresponding alcohol and carboxylic acid in a redox silent reaction with no observable change in A340. Ψ denotes the cofactor exchange step. The steps denoted by Ψ are insignificant during dismutation as cofactor remains enzyme bound throughout alternate oxidation and reduction (Alka, et al. 2010).
7
The Shaker family of Kv potassium channels has an modifications that have
not been reported to exist in Kv channels of prokaryotes and these adaptations
allow the Kv channel to perform functions that are unique to eukaryotic cells
(Long, et al. 2005). Long, et al. (2005) also reported that the β subunit had large
portals on the side of the structure, between the pore and cytoplasm, with
electrophysical properties which have a consistent result in similar studies on
electrophysiological inactivation gating and research postulates the potential of K+
channel regulation by the β subunit (Long, et al. 2005). Upon structural study of
the Kvβ2, it was reported that the subunit contains a similar sequence homology to
that of an aldo-keto reductase (AKR). This AKR fold allows the β subunit to
catalyse a redox reduction. The structural analysis also found that Kvβ2 has a
tightly bound nicotinamide cofactor; NAPDH. This bond is non covalent and the
region also contains an aldehyde binding site (Alka, K. et al. 2010; Weng, et al.
2006).
Crystal structure analysis of the ternary complex Kvβ2-NADPH-cortisone also
identified a binding site for cortisone on the proteins surface; supplementing the
binding site at the enzyme active site (Di Costanzo, et al. 2009). The AKR fold
previously mentioned, catalyses a redox reduction in this instance by reducing an
aldehyde to an alcohol via oxidation of the NADPH cofactor (Weng et al 2006).
Fig 1.2 shows the reaction as proposed by Alka, K., et al in 2010. A similar
scheme proposed by Weng, et al. (2006) also shows an AKR and enzyme binding
in sequence to an NADPH to form an Enzyme-NADPH-aldehyde complex. The
enzyme in this complex transfers a hydride from the NADPH cofactor to the
aldehyde thus producing an alcohol product which is followed suit by NADP+.
This is facilitated by AKRs having a higher affinity for NADPH over NADH
(Weng et al. 2006). The process in Fig.1.2 is reversible which allows the alcohol
to be oxidised to form an aldehyde and NADP+ to be converted to NADPH as
reported by Weng, et al in 2006. The rate of cofactor exchange in the above
process is however, slow thus indicating Kvβ is a slow enzyme. Weng, et al. also
shows in the 2006 publication that even though NADPH was oxidised over a two-
week period, there was still a presence of NADP+ in Kvβ; showing a tight
association which is to an extent due to a flexible loop which stretches over
NADPH and it’s binding site. It is this loop that possibly decelerates the
dissociation of the cofactor to a more prolonged period as reported. The reduction
8
of substrates such as 4-nitrobenzaldehyde (4-N-B-ald) is known to be catalysed by
Kvβ2 and the slow aldehyde-substrate dismutation has been shown through a
HPLC assay (Alka,, et al. 2010).
A potassium channel modulation function was reported as the bound cofactor,
NADPH, is oxidised. The regulation of channel activity, however questionable as
the adequate production of relative aldehydes may not be sufficient enough for the
channel regulation due to both enzymatic and non-enzymatic processes leading to
oxidative stress (Alka, et al. 2010). However, as noted above, this can be a slow
process but it is suspected that this redox reaction of the cofactor may be faster in
the presence of more specific physiological substrate which is yet to be elucidated
(Alka, et al. 2010). The rate of aldehyde reduction is linearly dependant on the
concentration of Kvβ2 and can be observed as a decrease in the peak area at the
450nm fluorescence peak (Alka, et al. 2010). Reports have shown that cortisone
promotes dissociation of the Kvβ2 from the K+ channel as it binds in two sites; at
the bound cofactor and the boundary of the Kvβ subunits and is known to not be a
substrate of the Kvβ2 protein, thus presenting the possibility of it being an
Fig.1.3: Structural features of Kvβ: A, structure of Kv1.2 (blue) in complex with Kvβ2 (red) in ribbon representation (Protein Data Bank code 2A79 (3)). The cell membrane is indicated by the straight lines.B, ribbon representation of Kvβ2 showing its structural fold (Protein Data Bank code 1QRQ (10)). The bound cofactor (cyan) and the conserved active site residues, Asp85, Tyr90, Lys118, and Asn158, are shown in stick representation. Residues Asp85 and Lys118 are labelled. A flexible loop that straddles the cofactor binding site is shown in yellow (Gulbis et al., 1999).
9
inhibitor (Alka, et al. 2010). This study looks at the inhibitory effect three pre-
identified potential inhibitors; Rutin, Quercitin and Resveratrol have on the
dismutation of aldehyde to form an alcohol product; 4-nitrobenzylalcohol (4-N-B-
alc). Cortisone is a steroid hormone and the three compounds mentioned
previously have been shown to have anti-inflammatory effects similar to those of
cortisone.
1.3 The Phenols: Rutin, Resveratrol and Quercitin
Rutin, Resveratrol and Quercitin are the three potential inhibitory compounds
of interest in this study. Fig.1.4 – 1.6 shows the phenolic structures of each
compound.
1.3.1 Quercitin and Rutin
Rutin is a primary flavanoid which can be found in a number of plants such as
Buckwheat. It is for this reason that there is high dietary consumption of Rutin as
buckwheat is used in the manufacture of noodles and rice (Koda, et al. 2008).
Rutin is a glycoside form of Quercitin of whose glycosides have free radical
scavenging activities (Andlauer, et al. 2001). As can be seen in Fig.1.4 and
Fig.1.6, Quercitin and Rutin are very similar in structure, therefore they have
similar mechanisms of action and biological affects. Rutin is a larger molecule
and has been shown to be less potent than Quercitin. This is possibly due to the
glycosylation adding a sterically-hindering group for inhibitory binding which
may impact, in the context of this study, at the interface binding site of the β
subunit in the Kvβ2-NADPH complex. Rutin is known to have an antioxidant
effect among various other biological effects which have a positive impact on
human health such as anti-inflammatory and a gastro protective effect due to its
augmentation of the antioxidant activity on the activity of glutathione peroxidase;
a selenoprotein that is recently being studied to link changes or abnormalities in
the protein with the etiology of some cancers, CVD, autoimmune disease and
diabetes (Lei, et al. 2007). The 2008 study by Koda, et al. investigated the
therapeutic effect of Rutin in reducing brain damage if administered per-orally in
rats. Koda, et al. showed that dietary supplementation of Rutin over a prolonged
period reversed the induced spatial memory impairment by trimethyltin. Other
health benefits such as chemopreventive activity was proposed by Andlauer, et al.
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with a requirement that intestinal absorptive-uptake must be carried out for this
link to be true contravening studies suggesting that Quercitin glycosides were
excreted rather than absorbed in human intestinal (Caco-2) cells (Andlauer, et al.
2001).
1.3.2 Resveratrol
Resveratrol is a phytoalexin that can be derived from the skin of fruits and I in
particular, red grapes among 70 other plant species. This attributes to high
concentrations of Resveratrol in red wine; approximately 50-100µg per gram of
grape skin (Athar, et al 2007). Like Rutin, Resveratrol is reported to have
antioxidant effects, potent anti-inflammatory and inhibition of the growth of
various cancer cells. Resveratrol is a compound that has helped spark an interest
in naturally occurring compounds being used as chemopreventives in human
cancers as it has been shown to have an effect on the tumour initiation, promotion
and progression stages of carcinogenisis (Athar, et al. 2007). Athar has also
reported that it is thought that Resveratrol can induce apoptosis of cells and
modulate cell growth pathways through its antioxidant activity.
Resveratrol, like other polyphenols can undergo glycosylation which has a
protective effect on Resveratrol by preventing it being degraded by oxidation thus
making it more stable and soluble and more soluble which is advantageous in the
gastrointestinal tract. It is this attribute that makes Resveratrol absorb more
efficiently than other polyphenols like Quercitin (Athar, et al. 2007). A review by
Athar, et al. in 2007 has shown that various administration routes have shown
positive outcomes when Resveratrol has been used in vivo against various
inhibited cancers in mice. Topical Resveratrol was tested in vivo for anti-
carcinogenisis activity on subcutaneous (SC) in the respect of non-melanoma skin
cancer and was proved to significantly reduced the prevalence of ultraviolet-B
(UVB)-mediated photo-toxicity at a topical dosage of 25µmol in SKH-1 hairless
mice and Soleas et al. identified in a 2002 a 60% reduction in papillomas when
Resveratrol was applied topically (Athar, et al. 2007). Modulation by the proteins
that regulate cell cycle have been associated to the anti-proliferative affects of
Resveratrol in such instances (Regan –Shaw, et al. 2004).
A comparison of red wine-consumers against other beverages showed that
there was a lower incidence of lung cancer among the subjects who consumed red
11
wine; associated with the high concentration of Resveratrol in red wine. Berge et
al. (2004) reported that Resveratrol inhibits the production of diol-epoxides;
compounds that have the potential to form covalent adducts with DNA and cause
structural alterations with mutations (Berge, et al. 2004). Studies on a number of
cancer types have shown Resveratrol to induce apoptosis in the carcinogenic cells,
also inhibit cell growth and significantly reduce the incidence of tumours while
also delaying the onset of tumourigenisis, in multiple targets and in a non-toxic
dose (Athar, et al. 2007). However, the dose which Resveratrol presents itself in
with red wine suggests that for health benefit to be observed by its administration
it may require synergistic combinations with compounds such as Quercitin and
ellagic acid; synergistic combinations which have been shown to induce apoptosis
in vitro and in vivo (Athar, et al. 2007). Resveratrol has been reported to have a
number of positive cardiogenic effects such as the reduction in incidences of
CVD. Its cardiogenic effects have been shown in the lowering of hyper / hypo-
tension clinical issues. This is thought to be due to Resveratrol inducing the
expression of endothelial nitric oxide synthase which is the enzyme responsible
for producing the vaso-dilating nitric oxides and decreasing the expression of the
endothelin-1; a vasoconstrictor (Das, et al. 2010). The endothelial cell is also
responsible for regulating the balance of endothelin-1 and nitric oxide which are
both important vasoconstrictors and vasodilators respectively; a function that
provides thromboresistance is shown to preclude atherogenesis (Das, et al. 2010).
Pharmacological intervention in cardiovascular medicine may be entering a new
age due to the range of health affects potentiated by Resveratrol such as cardiac
regeneration and the generation of autophagy (Das, et al. 2010).
1.4 Aldo-Keto Reductases
Aldo-keto reductases (AKR) form a large part of the cytosolic monomeric
NADPH-dependant carbonyl oxidoreductases along side another type of
oxidoreductase; short-chain dehydrogenase reductases (SDR) (Di Costanzo, et al.
2009). The AKR6A subfamily is associated with the Kvβ1-3 proteins which form
an (α/β)8-Barrell fold which links it structurally with AKRs, albeit with having a
low amino acid sequence similarity with other affiliates of the AKR group (Di
Costanzo, et al. 2009). The AKR family are known to reduce aldehydes and
ketones to their corresponding 1o and 2o alcohols and can be found in both
12
eukaryotes and prokaryotes (Penning and Drury, 2007). AKRs have also been
shown by Penning and Drury (2007) to reduce liophilic substrates such as
ketosteroids and retinals, thus regulating ligand access to nuclear receptors.
Substrate specificity of the AKR1 enzyme has been shown by Tipparaju et al.
(2008) to favour aromatic aldehydes, which contain electron withdrawing groups
in the para position of the aromatic ring or compounds that contain carbonyl
groups that are polarised by α,β- un-saturation, over cortisone which has shown
no activity. AKR also functions as an aldehyde reductase may have activity in
glucose metabolism and electron transport (Hyndman, et al. 2003). As mentioned
previously, cortisone binding sites were identified on the surface of the Kvβ-
NADPH-cortisone ternary complex, at which the cortisone binds backwards
compared to its binding profile within the active site of AKR1D1; a 5β-reductase,
which gives productive binding of progesterone as well as cortisone (Di Costanzo,
et al. 2009). Similar assessment of this structure has uncovered the link between
the binding of cortisone, in this fashion, to the surface of the protein and the
dissociation of β subunits from the Shaker potassium channel (Pan, et al. 2008).
AKRs are known to catalyse a number of reductions. In the bisequential
mechanism, in which reduction occurs in a central complex, binding of the
cofactor, NAPDH, supersedes the binding of a carbonyl substrate and is followed
by a release of the alcohol product and NADP+ in this respective order (Penning
and Drury, 2007). Penning and Drury (2007) also note that the AKRs rate
determining step can vary due to enzyme variation, i.e. most AKR reaction will
depend on the enzyme and rate of cofactor release. This is poignant in the regard
of Kvβ2 which has previously been noted by the author as being a slow reactive
protein thus having a slow rate of cofactor hydride transfer.
A link has been publicized about the implication of AKR in human diseases
such as diabetes. Catalysis of glucose conversion into sorbitol has been reported
as a role of aldose redcuatse; a prototypic member of the AKR family. This
conversion is the first step in the polyol pathway; a pathway which can occur in
the presence of chronic hyperglycaemia thus leading to diabetic complications
such as cataracts, retinopathy and nephropathy (Chang, et al. 2007).
13
Fig.1.4: Structure of Rutin (2-(3,4-dihydroxyphenyl)-4,5-dihydroxy-3[3,4,5-trihydoxy-6-[(3,4,5-trihydroxy-6methyl-oxan-2-yl)oxymethyl]oxan-2-yl]oxy-chromen-7-one)
Fig.1.5: Structure of Resveratrol (trans-3,4’,5-trihydroxystilbene
Fig.1.6: Chemical Structure of Quercitin
14
Chapter 2:
Materials & Methods
15
2.0 Materials and Methods
2.1 Materials
Materials are listed per company used for their respective acquisition.