Structural and Functional Insights into GID/CTLH E3 Ligase Complexes.

Multi-subunit E3 ligases facilitate ubiquitin transfer by coordinating various substrate receptor subunits with a single catalytic center. Small molecules inducing targeted protein degradation have exploited such complexes, proving successful as therapeutics against previously undruggable targets. The C-terminal to LisH (CTLH) complex, also called the glucose-induced degradation deficient (GID) complex, is a multi-subunit E3 ligase complex highly conserved from Saccharomyces cerevisiae to humans, with roles in fundamental pathways controlling homeostasis and development in several species. However, we are only beginning to understand its mechanistic basis. Here, we review the literature of the CTLH complex from all organisms and place previous findings on individual subunits into context with recent breakthroughs on its structure and function.

Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition.

The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/C(CORT), a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C).

The Cdk5 activator P39 specifically links muskelin to myosin II and regulates stress fiber formation and actin organization in lens.

Cyclin dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, requires p39 for its enzymatic activity, and is implicated in cytoskeletal organization and contraction in numerous cell types. The C-terminus of p39 binds muskelin, a multi-domain scaffolding protein known to affect cytoskeletal organization, but the mechanisms by which muskelin affects cytoskeletal organization remain unclear. The present study sought to determine whether p39 might serve as an adaptor protein that links muskelin to stress fibers and to investigate the possible biological relevance of such an interaction. Double immunoprecipitation showed that muskelin, p39, and myosin II are components of a single intracellular complex, and suppressing p39 abrogated the interaction between muskelin and the myosin subunits, demonstrating that p39 is required to link muskelin to myosin II. Muskelin is colocalized with myosin regulatory light chain (MRLC) and on stress fibers. The suppression of muskelin reduced Rho-GTP, MRLC phosphorylation, disrupted stress fiber organization, and promoted cell migration, all of which closely mimic the effect of Cdk5 inhibition. Moreover, suppressing muskelin and inhibiting Cdk5 together have no additional effect, indicating that muskelin plays an important role in Cdk5-dependent signaling. p39 is necessary and sufficient for Cdk5-dependent regulation of MRLC phosphorylation, as suppression of p39, but not p35, reduces MRLC phosphorylation. Together, these results demonstrate that p39 specifically links muskelin to myosin II and consequently, to stress fibers and reveal a novel role for muskelin in regulating myosin phosphorylation and cytoskeletal organization.

Structure of mouse muskelin discoidin domain and biochemical characterization of its self-association.

Muskelin is an intracellular kelch-repeat protein comprised of discoidin, LisH, CTLH and kelch-repeat domains. It is involved in cell adhesion and the regulation of cytoskeleton dynamics as well as being a component of a putative E3 ligase complex. Here, the first crystal structure of mouse muskelin discoidin domain (MK-DD) is reported at 1.55 Šresolution, which reveals a distorted eight-stranded ß-barrel with two short α-helices at one end of the barrel. Interestingly, the N- and C-termini are not linked by the disulfide bonds found in other eukaryotic discoidin structures. A highly conserved MIND motif appears to be the determinant for MK-DD specific interaction together with the spike loops. Analysis of interdomain interaction shows that MK-DD binds the kelch-repeat domain directly and that this interaction depends on the presence of the LisH domain.

Muskelin Coordinates PrPC Lysosome versus Exosome Targeting and Impacts Prion Disease Progression.

Cellular prion protein (PrPC) modulates cell adhesion and signaling in the brain. Conversion to its infectious isoform causes neurodegeneration, including Creutzfeldt-Jakob disease in humans. PrPC undergoes rapid plasma membrane turnover and extracellular release via exosomes. However, the intracellular transport of PrPC and its potential impact on prion disease progression is barely understood. Here we identify critical components of PrPC trafficking that also link intracellular and extracellular PrPC turnover. PrPC associates with muskelin, dynein, and KIF5C at transport vesicles. Notably, muskelin coordinates bidirectional PrPC transport and facilitates lysosomal degradation over exosomal PrPC release. Muskelin gene knockout consequently causes PrPC accumulation at the neuronal surface and on secreted exosomes. Moreover, prion disease onset is accelerated following injection of pathogenic prions into muskelin knockout mice. Our data identify an essential checkpoint in PrPC turnover. They propose a novel connection between neuronal intracellular lysosome targeting and extracellular exosome trafficking, relevant to the pathogenesis of neurodegenerative conditions.