BOR STEM Initiative at West Georgia: Our Story

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STEM II Initiative at the University of West Georgia

We will describe a BOR-funded program to enhance the STEM disciplines. We designed two approaches to support freshmen in STEM majors. The first is a bridge program focused on enhancing preparation for STEM coursework and college life. The second is an interdisciplinary seminar course designed to provide information about careers in STEM disciplines, including teaching in middle and high schools

Thermal Stress Induced Aggregation of Aquaporin 0 (AQP0) and Protection by α-Crystallin <i>via</i> Its Chaperone Function

<div><p>Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight) ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin) in close proximity to the lens membrane is physiologically relevant.</p></div

Chaperone-like activity of human α-crystallin gene products against target proteins.

<p><b>A</b>: (•) Recombinant human αA-crystallin (0.1 mg/ml) (homooligomer) and (▴) recombinant human αB-crystallin (0.1 mg/ml) (homooligomer), against (▪) thermal aggregation of ξ-crystallin (0.1 mg/ml) at 43°C. <b>B</b>: (•) Recombinant human αA-crystallin (0.2 mg/ml) (homooligomer) and (▴) recombinant human αB-crystallin (0.2 mg/ml) (homooligomer), against (▪) DTT-induced aggregation of insulin (0.2 mg/ml) at 43°C.</p

Chaperone-like activity of reconstituted human α-crystallin and its gene-products.

<p>Thermal aggregation of (a) AQP0 (0.15 mg/ml) at 49°C, (b) in presence of recombinant human αB- crystalline (0.15 mg/ml) (homooligomer), (c) recombinant human αA-crystallin (0.15 mg/ml) (homooligomer), and (d) in presence of recombinant human α-crystallin (0.15 mg/ml) (heterooligomer of αA- and αB-crystallins at 3∶1 ratio).</p

Effect of post-translational modifications and C-terminus truncation of AQP0 on thermal aggregation and chaperone function of α–crystallin.

<p>A: Effect of glycation of AQP0 on thermal aggregation and chaperone function. Lens membranes were incubated with 1 M glucose for 4 days. Glycated AQP0 was solubilized in 1% octyl glucoside and subjected to thermal stress. a - glycated AQP0; b - non-glycated AQP0; c - glycated AQP0 + α–crystallin; d - non-glycated AQP0 + α–crystallin. Inset: SDS-PAGE of <i>in vitro</i> glycated AQP0. M – Markers; 1 – Control incubation; 2 - 1 M glucose incubation. B<i>:</i> Effect of C-terminus truncation of AQP0 on thermal aggregation and chaperone function: Lens membranes were treated with Glu-C protease and the resultant C-terminus truncated AQP0 was solubilized in 1% octyl glucoside, and then subjected to thermal stress. a - control AQP0; b - C-terminus truncated AQP0; c - control AQP0 + α-crystallin; d - C-terminus truncated AQP0 + α-crystallin. Inset: SDS-PAGE of C-terminus truncated AQP0. 1 – Control AQP0; 2 – Glu-C protease digested AQP0; M – Markers.</p

HPLC temporal profile of thermally stressed AQP0.

<p>A: Samples of OG solubilized calf AQP0 (0.15 mg/ml in phosphate buffer, pH 7.4 containing 100 mM NaCl and 1% OG) were incubated at 50°C for various time intervals. Note the AQP0 monomer resolved as a major peak at 26 kDa protein and the AQP0 aggregate at ∼ 1000 kDa (see 3 min at 50°C). When the AQP0 was maintained at 50°C for 6 min or more, these AQP0 aggregates also disappeared. B: Addition of α-crystallin in the incubation mixture resulted in the formation of α-crystallin and AQP0 complexes resolved at ∼800 kDa.</p

Thermal aggregation of AQP0 and protection by α-crystallin.

<p>Detergent solubilized AQP0 (0.15 mg/ml in 10 mM phosphate buffer, pH 7.4 containing 1% OG) was subjected to thermal stress by gradually increasing the temperature and the absorbance monitored at 360 nm on a Jasco 715 Spectropolarimeter.</p

Gel electrophoresis analysis of thermal-denaturation of AQP0 and protection by α-crystallin.

<p>Detergent solubilized AQP0 (0.15 mg/ml in 10 mM phosphate buffer, pH 7.4 containing 1% OG) was subjected to thermal stress in a thermal cycler (5 min at each given temperature) without and with α-crystallin and resolved on 12% SDS-PAGE. Left panel - AQP0 alone and Right panel - AQP0 + α-crystallin. MW – molecular weight markers (range 20-200 kDa); lane 1 – 25°C; lane 2 – 30°C; lane 3 – 35°C; lane 4 – 40°C; lane 5 – 45°C; lane 6 – 50°C; lane 7 – 55°C and lane 8 – 60°C.</p

Secondary structural changes of AQP0 during thermal stress and protection by α–crystallin.

<p>A<i>:</i> Detergent solubilized AQP0 (0.1 mg/ml in 10 mM phosphate buffer, pH 7.4 containing 100 mM NaCl and 1% OG) was subjected to thermal stress for 5 min at a given temperature. Following the thermal stress, the protein solution was immediately cooled to 25°C and Far UV CD studies were done at 25°C using a circular dichroism spectropolarimeter. Note progressive loss of α-helical structures with increasing temperature and these changes are irreversible. B<i>:</i> The same assay was done in the presence of bovine α–crystallin (at a ratio of 1∶1 weight/weight).</p