Human V-ATPase a-subunit isoforms bind specifically to distinct phosphoinositide phospholipids.

Vacuolar H+-ATPases (V-ATPases) are highly conserved multisubunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue- and organelle-specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle-specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms, and we hypothesize that the aNT domains of these isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. We determined that bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP-binding sites, we identified potential binding sites in the HuaNT domains by sequence comparisons and existing subunit structures and models. We found that mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles.

Human V-ATPase a-subunit isoforms bind specifically to distinct phosphoinositide phospholipids.

V-ATPases are highly conserved multi-subunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue and organelle specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms. We hypothesize that the aNT domains of the human isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. Bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP binding sites, potential binding sites in the HuaNT domains were identified by sequence comparisons and existing subunit structures and models. Mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data also suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles.

Alzheimer's Disease Markers in Aged ApoE-PON1 Deficient Mice.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging. Cardiovascular risk factors like hypertension and atherosclerosis increase the risk for AD. Polymorphic alleles of apolipoprotein E (ApoE) are one of the main genetic determinants of AD. OBJECTIVE: Mice, double-knockout (DKO) for ApoE (major cholesterol carrier in brain) and PON1 (paroxonase1, reduces oxidative stress), showed severe age-dependent atherosclerosis of the arteries carrying blood to the brain even on normal diet. This prompted us to investigate the possibility of an AD pathology resulting from the deficiency of ApoE and the induction of oxidative stress. METHODS: Atherosclerotic lesions were quantified by ImageJ. The brain hippocampus of young and old ApoE-PON1 DKO mice and control mice were harvested. RT-PCR analysis was performed for mRNA levels of AD specific markers. Blood levels of S100 calcium-binding protein B (S100B) protein were measured by ELISA. H&E as well as immunostaining was performed to detect amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs) in brain tissues. Evans blue dye was used to evaluate the vascular permeability and blood-brain barrier (BBB) dysfunction. RESULTS: Results showed that the older DKO mice had severe carotid atherosclerosis, increased mRNA levels of AD markers in brain tissue, and elevated levels of serum S100B protein. Immunological staining confirmed the characteristics of AD. Ex-vivo imaging showed higher levels of Evans blue dye in the ApoE-PON1 DKO mice brain tissues, pointing toward impaired vasculature. CONCLUSION: Aged ApoE-PON1 DKO mice displaying AD specific markers along with Aß plaques, NFTs, and disrupted BBB suggests the animals are suffering from AD.

Alzheimer's Disease-Current Status and Future Directions.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder of the brain. The presence of amyloid-beta (Aß) plaques, neurofibrillary tangles (NFTs), loss of neurons, synapses, and altered sensory perceptions, including memory loss, delineate AD. However, the cause of AD is not clearly known. Several genetic and nongenetic factors have been implicated in the disease. Of the genes, the ɛ4 allele of apolipoprotein E is the largest known genetic risk factor of AD. This review article focuses on the various genetic and other predisposing factors that account for AD, pathophysiology of the disease, and the mechanisms by which Aß plaques and NFTs are formed and could affect AD brain. In addition, recent advances and current diagnostics available for AD patients are detailed. As oxidative stress has been implicated in the etiology of the disease, special emphasis is given for nutrition based antioxidant therapies and interventional strategies for reducing/treating AD.