Mutagenesis, Hydrogen-Deuterium Exchange, and Molecular Docking Investigations Establish the Dimeric Interface of Human Platelet-Type 12-Lipoxygenase.

It was previously shown that human platelet 12S-lipoxygenase (h12-LOX) exists as a dimer; however, the specific structure is unknown. In this study, we create a model of the dimer through a combination of computational methods, experimental mutagenesis, and hydrogen-deuterium exchange (HDX) investigations. Initially, Leu183 and Leu187 were replaced by negatively charged glutamate residues and neighboring aromatic residues were replaced with alanine residues (F174A/W176A/L183E/L187E/Y191A). This quintuple mutant disrupted both the hydrophobic and π-π interactions, generating an h12-LOX monomer. To refine the determinants for dimer formation further, the L183E/L187E mutant was generated and the equilibrium shifted mostly toward the monomer. We then submitted the predicted monomeric structure to protein-protein docking to create a model of the dimeric complex. A total of nine of the top 10 most energetically favorable docking conformations predict a TOP-to-TOP dimeric arrangement of h12-LOX, with the α-helices containing a Leu-rich region (L172, L183, L187, and L194), corroborating our experimental results showing the importance of these hydrophobic interactions for dimerization. This model was supported by HDX investigations that demonstrated the stabilization of four, non-overlapping peptides within helix α2 of the TOP subdomain for wt-h12-LOX, consistent with the dimer interface. Most importantly, our data reveal that the dimer and monomer of h12-LOX have distinct biochemical properties, suggesting that the structural changes due to dimerization have allosteric effects on active site catalysis and inhibitor binding.

Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences.

Knowledge about cervical cancer early warning signs and symptoms, risk factors and vaccination among students at a medical school in Al-Ahsa, Kingdom of Saudi Arabia.

BACKGROUND: Cervical cancer is the second most common cancer among females and also the most preventable. In the literature there is abundant evidence that awareness regarding cervical cancer and its prevention is low in the developing countries. Medical students are the future health professionals and can play an important role in increasing awareness among the general population. To assess the knowledge regarding symptoms, risk factors and prevention of cervical carcinoma among medical students in th Kingdom of Saudi Arabia, the present study was planned. MATERIALS AND METHODS: This cross-sectional study was conducted using a self-administered questionnaire with students at the College of Medicine, King Faisal University, Al-Ahsa, KSA, from December 2012 to May 2013. RESULTS: The responses of 188 students (males 111, females 77) in their second, third, fourth, and fifth years were recorded and used in the data analysis. The majority of the students were not aware of the early warning signs, symptoms and risk factors. On average, only 43.7% males and 56% of females were aware about the early signs and symptoms whereas 51.4% males and 57.8% females had knowledge about the risk factors of cervical cancers. Some 55% males and 46.8% females were unable to select the correct answer regarding human papilloma virus (HPV) infection as the cause of cervical cancer. Majority of the students (67%) were not aware about the availability of vaccine against HPV. CONCLUSIONS: Lack of knowledge regarding early signs and symptoms, risk factors and prevention of cervical cancer was observed in the present study.