A study on the two binding sites of hexokinase on brain mitochondria

<p>Abstract</p> <p>Background</p> <p>Type I hexokinase (HK-I) constitutes the predominant form of the enzyme in the brain, a major portion of which is associated with the outer mitochondrial membrane involving two sets of binding sites. In addition to the glucose-6-phosphate (G6P)-sensitive site (Type A), the enzyme is bound on a second set of sites (Type B) which are, while insensitive to G6P, totally releasable by use of high concentrations of chaotropic salts such as KSCN. Results obtained on release of HK-I from these "sites" suggested the possibility for the existence of distinct populations of the bound enzyme, differing in susceptibility to release by G6P.</p> <p>Results</p> <p>In the present study, the sensitivity of HK-I toward release by G6P (2 mM) and a low concentration of KSCN (45 mM) was investigated using rat brain, bovine brain and human brain mitochondria. Partial release from the G6P-insensitive site occurred without disruption of the mitochondrial membrane as a whole and as related to HK-I binding to the G6P-sensitive site. While, as expected, the sequential regime release-rebinding-release was observed on site A, no rebinding was detectable on site B, pre-treated with 45 mM KSCN. Also, no binding was detectable on mitochondria upon blocking site A for HK-I binding utilizing dicyclohexylcarbodiimide (DCCD), followed by subsequent treatment with KSCN. These observations while confirmed the previously-published results on the overall properties of the two sites, demonstrated for the first time that the reversible association of the enzyme on mitochondria is uniquely related to the Type A site.</p> <p>Conclusion</p> <p>Use of very low concentrations of KSCN at about 10% of the level previously reported to cause total release of HK-I from the G6P- insensitive site, caused partial release from this site in a reproducible manner. In contrast to site A, no rebinding of the enzyme takes place on site B, suggesting that site A is 'the only physiologically-important site in relation to the release-rebinding of the enzyme which occur in response to the energy requirements of the brain. Based on the results presented, a possible physiological role for distribution of the enzyme between the two sites on the mitochondrion is proposed.</p

Quantitative Measurement of the Affinity of Toxic and Nontoxic Misfolded Protein Oligomers for Lipid Bilayers and of its Modulation by Lipid Composition and Trodusquemine.

Many neurodegenerative diseases are associated with the self-assembly of peptides and proteins into fibrillar aggregates. Soluble misfolded oligomers formed during the aggregation process, or released by mature fibrils, play a relevant role in neurodegenerative processes through their interactions with neuronal membranes. However, the determinants of the cytotoxicity of these oligomers are still unclear. Here we used liposomes and toxic and nontoxic oligomers formed by the same protein to measure quantitatively the affinity of the two oligomeric species for lipid membranes. To this aim, we quantified the perturbation to the lipid membranes caused by the two oligomers by using the fluorescence quenching of two probes embedded in the polar and apolar regions of the lipid membranes and a well-defined protein-oligomer binding assay using fluorescently labeled oligomers to determine the Stern-Volmer and dissociation constants, respectively. With both approaches, we found that the toxic oligomers have a membrane affinity 20-25 times higher than that of nontoxic oligomers. Circular dichroism, intrinsic fluorescence, and FRET indicated that neither oligomer type changes its structure upon membrane interaction. Using liposomes enriched with trodusquemine, a potential small molecule drug known to penetrate lipid membranes and make them refractory to toxic oligomers, we found that the membrane affinity of the oligomers was remarkably lower. At protective concentrations of the small molecule, the binding of the oligomers to the lipid membranes was fully prevented. Furthermore, the affinity of the toxic oligomers for the lipid membranes was found to increase and slightly decrease with GM1 ganglioside and cholesterol content, respectively, indicating that physicochemical properties of lipid membranes modulate their affinity for misfolded oligomeric species

A genetic variant in CDKN2A/2B locus was associated with poor prognosis in patients with 1 esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is among the leading causes of cancer related death. Despite extensive efforts in identifying valid cancer prognostic biomarkers, only a very small number of markers have been identified. Several genetic variants in the 9p21 region have been identified that are associated with the risk of multiple cancers. Here, we explored the association of two genetic variants in the 9p21 region, CDKN2A/B, rs10811661 and rs1333049 for the first time in 273 subjects with, or without ESCC. We observed that patients with ESCC had a higher frequency of a TT genotype for rs10811661 than individuals in the control group, and this polymorphism was also associated with tumor size. Moreover, a CC genotype for the rs1333049 polymorphism was associated with a reduced OS of patients with ESCC. In particular, patients with a CC (rs1333049) genotype had a significantly shorter OS (CC genotype: 34.5±8.9 months vs. CG+GG: 47.7±5.9 months; p value= 0.03). We have also shown the association of a novel genetic variant in CDKN2B gene with clinical outcome of ESCC patients. Further investigations are warranted in a larger population to explore the value of emerging markers as a risk stratification marker in ESCC. Key word: Esophageal squamous cell carcinoma, risk marker, CDKN2A/B, polymorphis

Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein

Inhibition of amyloid fibril formation and cytotoxicity by a chemical analog of Curcumin as a stable inhibitor

In this paper, we endeavor for predicting the performance of quantized compressive sensing under the use of sparse reconstruction estimators. We assume that a high rate vector quantizer is used to encode the noisy compressive sensing measurement vector. Exploiting a block sparse source model, we use Gaussian mixture density for modeling the distribution of the source. This allows us to formulate an optimal rate allocation problem for the vector quantizer. Considering noisy CS quantized measurements, we analyze upper- and lower-bounds on reconstruction error performance guarantee of two estimators - convex relaxation based basis pursuit de-noising estimator and an oracle-assisted least-squares estimator.QC 20130118</p

Effect of Cinnamomum Verum Extract on the Amyloid Formation of Hen Egg-white Lysozyme and Study of its Possible Role in Alzheimer’s Disease

Introduction: Diagnosing and treating diseases associated with amyloid fibers remain a great challenge despite of intensive research carried out. One important approach in the development of therapeutics is the use of herbal extracts which are rich in aromatic small molecules. Cinnamomum verum extract (CE) contains proanthocyanidin and cinnamaldehyde, which have been suggested to be capable of directly inhibiting amyloid fibril formation in vitro. This study is aimed at characterizing the inhibitory activity of CE against the fibrillation of hen egg white lysozyme (HEWL). Methods: Acidic pH and high temperatures were used to drive the protein towards amyloid formation. Lysozyme was dissolved at 2 mg/mL in 50mM glycine buffer (pH 2.5), and then incubated at 57 °C for the specified durations while stirred gently by Teflon magnetic bars. Various techniques including thioflavin T, fluorescence, Congo red absorbance assay and AFM micrography were used to characterize the HEWL fibrillation processes. Results: In the absence of CE typical amyloid fibrils (like amyloids formed in Alzheimer disease) became evident after 48 h of incubation. Upon incubation with various extract concentrations in the range of 0.1-1 mg/ml, formation of fibrillar assemblies were significantly inhibited (p<0.05). AFM analysis and MTT assay also confirmed the role of the extract in amyloid inhibition. Our studies showed that the presence of CE did not have any effect on protein stabilization and thus directly interact with amyloid structure and inhibit formation of these structures. Furthermore, a docking experiment showed that a pi-pi interaction may occur between the aromatic component of cinnamaldehyde and W62. Interestingly, W62 is one of the principal aromatic residues that interact with glycine amide, which is an aggregation suppressor of HEWL. Discussion: These observations suggest that aromatic small molecules of CE may directly insert into amyloidogenic core of early aggregates and inhibit amyloid fibril formation by disrupting the pi-pi interactions

Silver nano particles ameliorate learning and spatial memory of male Wistar rats by prevention of amyloid fibril-induced neurotoxicity

Alzheimer's disease (AD) is a chronic degenerative disease characterized by the presence of amyloid plaques and neurofibrillary tangles (NFTs), which results into memory and learning impairments. In the present study, we showed that the aggregates formed by a protein that has no link with Alzheimer's disease, namely the hen egg white lysozyme (HEWL), were cytotoxic and decreased spatial learning and memory in rats. The effect of Ag-nano particles (Ag-NPs) was investigated on disruption of amyloid aggregation and preservation of cognitive behavior of rats. Twenty-four male Wistar rats were divided into 4 groups including a control group, and injected with either scopolamine, lysozyme or aggregates pre-incubated with Ag-NPs. Rats' behavior was monitored using Morris water maze (MWM) twenty days after injections. HEWL aggregation in the presence and absence of the Ag-NPs was assayed by Thioflavin T binding, atomic force microscopy and cell-based cytotoxicity assay. Ag-NPs were capable to directly disrupt HEWL oligomerization and the resulting aggregates were non-toxic. We also showed that rats of the Ag-NPs group found MWM test platform in less time and with less distance traveled, in comparison with lysozyme group. Ag-NPs also increased the percentage of time elapsed and the distance swum in the target quadrant in the rat model of AD, in probe test. These observations suggest that Ag-NPs improved spatial learning and memory by inhibiting amyloid fibril-induced neurotoxicity. Furthermore, we suggest using model proteins as a valid tool to investigate the pathogenesis of Alzheimer's disease. PMID: 2922086