Structural features and oligomeric nature of human podocin domain

Podocytes are crucial cells of the glomerular filtration unit and plays a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and charge-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101–125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126–350). We show that the podocin domain majorly homo-oligomerizes into a 16-mer. Circular dichroism and fluorescence spectroscopy suggest that the 16-mer oligomer has considerable secondary structure and moderate tertiary packing.     

Tyrosinase-like activity and estradiol binding in rat uterine nuclear extracts.

Nuclear extracts from the uteri of estradiol-implanted rats contain a tyrosinase-like enzyme that has three activities: monophenolase or cresolase, diphenolase or catecholase, and estrogen binding. When [3H]estradiol was used as a substrate, 3H2O was released from the A ring in the presence of copper and ascorbic acid. The optimal concentrations of these cofactors for the cresolase activity were established. The cresolase activity was lost on attempts at further purification. Estradiol binding was observed in conjunction with the enzymatic activity and was dependent on the presence of ascorbic acid and copper. The most potent inhibitors of 3H2O release from [3H]estradiol were those with a dihydroxyphenol moiety. The reaction was also sensitive to sulfhydryl reagents. These features of the enzyme are distinctive from other oxidases capable of attacking the aromatic ring of estrogens.     

Tyrosinase-like polypeptides in the uterus and in the central nervous system of rats.

Nuclear and cytosolic fractions of rat uteri and tissues from the central nervous system contain proteins that are recognized by a polyclonal tyrosinase antibody. This antibody eliminates the cresolase activity of uterine nuclear extract when estradiol is used as substrate. Thus, it appears that tyrosinase-like proteins might be present in tissues not generally considered to chain such an enzyme.     

Concerning the structure of apoE

Apolipoprotein E (apoE), first described in 1973, is a truly fascinating protein. While studies initially focused on its role in cholesterol and lipid metabolism, one apoE isoform (apoE4) is a major risk factor for development of late onset Alzheimer's disease. Yet the difference between apoE3, the common form, and apoE4 is a single amino acid of the 299 in this 34 kDa protein. Structure determination of the two domain full length apoE3 protein was only accomplished in 2011 and supports the notion that mutations in the N‐terminal domain can be propagated through the structure to the C‐terminal domain. Understanding the structural differences between apoE3 and apoE4 is critical for finding ways to modulate the deleterious effect of apoE4.     

Detecting Amyloid-β Aggregation with Fiber-Based Fluorescence Correlation Spectroscopy

Soluble aggregates critically influence the chemical and biological aspects of amyloid protein aggregation, but their population is difficult to measure, especially in vivo. We take an optical fiber-based fluorescence correlation spectroscopy (FCS) approach to characterize a solution of aggregating amyloid-β molecules. We find that this technique can easily resolve aggregate particles of size 100 nm or greater in vitro, and the size distribution of these particles agrees well with that obtained by conventional FCS techniques. We propose fiber FCS as a tool for studying aggregation in vivo.