Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures.

Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300-600nm in length. These were clearly different from annular oligomers 120-180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.

Imperfect interface of Beclin1 coiled-coil domain regulates homodimer and heterodimer formation with Atg14L and UVRAG.

Beclin 1 is a core component of the Class III Phosphatidylinositol 3-Kinase VPS34 complex. The coiled coil domain of Beclin 1 serves as an interaction platform for assembly of distinct Atg14L- and UVRAG-containing complexes to modulate VPS34 activity. Here we report the crystal structure of the coiled coil domain that forms an antiparallel dimer and is rendered metastable by a series of 'imperfect' a-d' pairings at its coiled coil interface. Atg14L and UVRAG promote the transition of metastable homodimeric Beclin 1 to heterodimeric Beclin1-Atg14L/UVRAG assembly. Beclin 1 mutants with their 'imperfect' a-d' pairings modified to enhance self-interaction, show distinctively altered interactions with Atg14L or UVRAG. These results suggest that specific utilization of the dimer interface and modulation of the homodimer-heterodimer transition by Beclin 1-interacting partners may underlie the molecular mechanism that controls the formation of various Beclin1-VPS34 subcomplexes to exert their effect on an array of VPS34-related activities, including autophagy.

The Beclin 1-VPS34 complex--at the crossroads of autophagy and beyond.

An increasing body of research on autophagy provides overwhelming evidence for its connection to diverse biological functions and human diseases. Beclin 1, the first mammalian autophagy protein to be described, appears to act as a nexus point between autophagy, endosomal, and perhaps also cell death pathways. Beclin 1 performs these roles as part of a core complex that contains vacuolar sorting protein 34 (VPS34), a class III phosphatidylinositol-3 kinase. The precise mechanism of Beclin 1-mediated regulation of these cellular functions is unclear, but substantial progress has recently been made in identifying new players and their functions in Beclin 1-VSP34 complexes. Here we review emerging studies that are beginning to unveil the physiological functions of Beclin 1-VPS34 in the central control of autophagic activity and other trafficking events through the formation of distinct Beclin 1-VPS34 protein complexes.

Cell "self-eating" (autophagy) mechanism in Alzheimer's disease.

The autophagy pathway is the major degradation pathway of the cell for long-lived proteins and organelles. Dysfunction of autophagy has been linked to several neurodegenerative disorders that are associated with an accumulation of misfolded protein aggregates. Alzheimer's disease, the most common neurodegenerative disorder, is characterized by 2 aggregate forms, tau tangles and amyloid-beta plaques. Autophagy has been linked to Alzheimer's disease pathogenesis through its merger with the endosomal-lysosomal system, which has been shown to play a role in the formation of the latter amyloid-beta plaques. However, the precise role of autophagy in Alzheimer's disease pathogenesis is still under contention. One hypothesis is that aberrant autophagy induction results in an accumulation of autophagic vacuoles containing amyloid-beta and the components necessary for its generation, whereas other evidence points to impaired autophagic clearance or even an overall reduction in autophagic activity playing a role in Alzheimer's disease pathogenesis. In this review, we discuss the current evidence linking autophagy to Alzheimer's disease as well as the uncertainty over the exact role and level of autophagic regulation in the pathogenic mechanism of Alzheimer's disease.

Specific N-terminal mutations in the human androgen receptor induce cytotoxicity.

Polyglutamine (polyQ) stretch amplification in different proteins causes neurodegenerative disease. These proteins form intracellular aggregates thought to be cytotoxic but differ in pathology and tissue specificity. Here, we demonstrate that specific sequences outside the polyQ stretch of the human androgen receptor contribute to polyQ pathology. An exchange of two N-terminal serine phosphorylation residues to alanine in the wild type androgen receptor (ARQ22dm) resulted in cytoplasmic accumulation and increased early hormone-dependent aggregation of the receptor. In a Drosophila model, the ARQ22dm was cytotoxic, and developing larvae expressing this receptor showed behavioral abnormalities and severely impaired locomotion. In contrast, the same double mutation in an androgen receptor with an extended polyQ stretch was less toxic. The response of the receptors to inhibitors of polyglutamine toxicity is altered by the amino acid exchanges suggesting that careful consideration is needed in the choice of potential therapies of disorders involving toxic polyQ species.

Receptor internalization-independent activation of Smad2 in activin signaling.

Activin, a member of the TGFbeta family of cytokines, signals through heteromeric transmembrane complexes composed of type I and type II Ser/Thr kinase receptors. Activated by type II receptors, the type I receptor phosphorylates, thereby activating its effectors Smad2 and Smad3. It has been shown that the ligand-bound TGFbeta receptors endocytose to early endosomes, where they phosphorylate Smads. However, whether TGFbeta and activin can signal without receptor internalization is still in question. We report that a mutation changing Trp477 to Ala in the kinase domain rendered the type I activin receptor Alk4 unable to undergo ligand-dependent internalization. However, the resultant receptor, named Alk4W477A, retained the ability to phosphorylate Smad2 and mediate activin-induced transcription activation. Also, a Trp477 to Ala mutation abolished the endocytosis of Alk4T206D, a constitutively active type I activin receptor. The action of the mutant Alk4T206D became activin dependent. Finally, blocking endocytosis by depletion of intracellular potassium did not inhibit Smad2 phosphorylation by Alk4W477A. Taken together, our data indicate that activin receptors can transduce activin signals without endocytosis and suggest the possibility that an endocytosis-independent activin signaling pathway exists, which may act as an alternative mechanism for signal transduction.