Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates.

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington's disease.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.