"Trim"ming PolyQ proteins with engineered PML.

Protein abnormalities are the major cause of neurodegenerative diseases such as spinocerebellar ataxia (SCA). Protein misfolding and impaired degradation leads to the build-up of protein aggregates inside the cell, which may further cause cellular degeneration. Reducing levels of either the soluble misfolded form of the protein or its precipitated aggregate, even marginally, could significantly improve cellular health. Despite numerous pre-existing strategies to target these protein aggregates, there is considerable room to improve their specificity and efficiency. In this study, we demonstrated the enhanced intracellular degradation of both monomers and aggregates of mutant ataxin1 (Atxn1 82Q) by engineering an E3 ubiquitin ligase enzyme, promyelocytic leukemia protein (PML). Specifically, we showed enhanced degradation of both soluble and aggregated Atxn1 82Q in mammalian cells by targeting this protein using PML fused to single chain variable fragments (scFvs) specific for monomers and aggregates of the target protein. The ability to solubilize Atxn1 82Q aggregates was due to the PML-mediated enhanced SUMOylation of the target protein. This ability to reduce the intracellular levels of both misfolded forms of Atxn1 82Q may not only be useful for treating SCA, but also applicable for the treatment of other PolyQ disorders.

Understanding How Wnt Influences Destruction Complex Activity and ß-Catenin Dynamics.

Despite extensive research on the canonical Wnt signaling pathway, the mechanism by which this signal downregulates the activity of destruction complexes and inhibits ß-catenin degradation remains controversial. In particular, recent attention has focused on two main competing mechanisms-inhibition of phosphorylation and inhibition of ubiquitination. Our combined experimental and theoretical analysis demonstrates that the disassembly of a fraction of the intracellular destruction complexes results in the partial inhibition of both ß-catenin phosphorylation and ubiquitination. This inhibition is spatially patterned, consistent with the relocalization of some destruction complexes to the cellular membrane upon Wnt stimulation. Moreover, in contrast to the generally accepted view that the destruction complex is highly processive, our analysis supports a distributive model, in which ß-catenin can dissociate from the complex between sequential phosphorylation events. Understanding the fundamental mechanism by which Wnt signaling is regulated provides a rational basis for tuning the pathway for scientific and therapeutic purposes.

Dissecting the structural and functional features of the Luteinizing hormone receptor using receptor specific single chain fragment variables.

The Luteinizing hormone receptor (LHR) has a large extracellular domain (amino acid residues, a.a.1-355) and a transmembrane domain (TMD; a.a. 356-699), essential for hormone binding and signaling, respectively. The LHR hinge region (a.a. 256-355) connects the two domains and acts as an activating switch for the receptor by an unknown mechanism. LHR hinge-specific Single chain fragment variables (ScFv) stimulated cAMP production by the stable and transiently transfected cell lines expressing LHR in a hormone-independent manner and the C-terminal region of LHR hinge (a.a. 313-349) was identified as the probable epitope for one agonistic ScFv. This epitope attained a helical conformation upon agonistic ScFv binding and the activity of the ScFv was dependent on Y331 sulfation. ScFv was also able to activate TMD mutants, D578Y and A593P, reemphasizing the role of TM helix VI in LHR activation.

Biodegradable and biocompatible polyampholyte microgels derived from chitosan, carboxymethyl cellulose and modified methyl cellulose.

Two biocompatible and biodegradable polyampholyte microgels, namely chitosan-carboxymethyl cellulose (CS-CMC) and chitosan-modified methyl cellulose (CS-ModMC) were synthesized by an inverse microemulsion technique. The CS-CMC microgel system was pH-responsive while the CS-ModMC system possessed both pH and thermo-responsive properties. For CS-CMC system, the number of -OCH2COOH and -NH2 groups was determined to be 1.5 and 1.1meq/g of microgel, respectively. In the pH range of 4-9, the zeta potential values varied from +10 to -40mV, while the hydrodynamic radius varied from 160nm in the swollen state (acidic and basic pH) to 110nm in the "collapse" state (neutral pH). Furthermore, TEM micrographs confirmed the swelling/deswelling behaviour of CS-CMC microgel particles at acidic, neutral and basic conditions. For CS-ModMC system, the number of -OCH2COOH and -NH2 groups was determined to be 0.8 and 0.6meq/g microgel, respectively. In the pH range of 4-9, the surface charge on the microgels varied from +25 to -60mV and the hydrodynamic radii were 190nm at low pH, 80nm at neutral pH, to 120nm at a high pH. In vitro drug release studies confirmed that CS-CMC microgels could encapsulate and release a model drug, thus they could potentially be used as biocompatible and biodegradable drug carriers.