The Sphinx and the egg: Evolutionary enigmas of the (glyco)sphingolipid biosynthetic pathway.

In eukaryotes, the de novo synthesis of sphingolipids (SLs) consists of multiple sequential steps which are compartmentalized between the endoplasmic reticulum and the Golgi apparatus. Studies over many decades have identified the enzymes in the pathway, their localization, topology and an array of regulatory mechanisms. However, little is known about the evolutionary forces that underly the generation of this complex pathway or of its anteome, i.e., the metabolic pathways that converge on the SL biosynthetic pathway and are essential for its activity. After briefly describing the pathway, we discuss the mechanisms by which the enzymes of the SL biosynthetic pathway are targeted to their different subcellular locations, how the pathway per se may have evolved, including its compartmentalization, and the relationship of the pathway to eukaryogenesis. We discuss the circular interdependence of the evolution of the SL pathway, and comment on whether current Darwinian evolutionary models are able to provide genuine mechanistic insight into how the pathway came into being.

The C-Terminus of the PSMA3 Proteasome Subunit Preferentially Traps Intrinsically Disordered Proteins for Degradation.

The degradation of intrinsically disordered proteins (IDPs) by a non-26S proteasome process does not require proteasomal targeting by polyubiquitin. However, whether and how IDPs are recognized by the non-26S proteasome, including the 20S complex, remains unknown. Analyses of protein interactome datasets revealed that the 20S proteasome subunit, PSMA3, preferentially interacts with many IDPs. In vivo and cell-free experiments revealed that the C-terminus of PSMA3, a 69-amino-acids-long fragment, is an IDP trapper. A recombinant trapper is sufficient to interact with many IDPs, and blocks IDP degradation in vitro by the 20S proteasome, possibly by competing with the native trapper. In addition, over a third of the PSMA3 trapper-binding proteins have previously been identified as 20S proteasome substrates and, based on published datasets, many of the trapper-binding proteins are associated with the intracellular proteasomes. The PSMA3-trapped IDPs that are proteasome substrates have the unique features previously recognized as characteristic 20S proteasome substrates in vitro. We propose a model whereby the PSMA3 C-terminal region traps a subset of IDPs to facilitate their proteasomal degradation.

Oncogenic addiction to high 26S proteasome level.

Proteasomes are large intracellular complexes responsible for the degradation of cellular proteins. The altered protein homeostasis of cancer cells results in increased dependency on proteasome function. The cellular proteasome composition comprises the 20S catalytic complex that is frequently capped with the 19S regulatory particle in forming the 26S proteasome. Proteasome inhibitors target the catalytic barrel (20S) and thus this inhibition does not allow the deconvolution of the distinct roles of 20S versus 26S proteasomes in cancer progression. We examined the degree of dependency of cancer cells specifically to the level of the 26S proteasome complex. Oncogenic transformation of human and mouse immortalized cells with mutant Ras induced a strong posttranscriptional increase of the 26S proteasome subunits, giving rise to high 26S complex levels. Depletion of a single subunit of the 19S RP was sufficient to reduce the 26S proteasome level and lower the cellular 26S/20S ratio. Under this condition the viability of the Ras-transformed MCF10A cells was severely compromised. This observation led us to hypothesize that cancer cell survival is dependent on maximal utilization of its 26S proteasomes. We validated this possibility in a large number of cancer cell lines and found that partial reduction of the 26S proteasome level impairs viability in all cancer cells examined and was not correlated with cell doubling time or reduction efficiency. Interstingly, normal human fibroblasts are refractory to the same type of 26S proteasome reduction. The suppression of 26S proteasomes in cancer cells activated the UPR and caspase-3 and cells stained positive with Annexin V. In addition, suppression of the 26S proteasome resulted in cellular proteasome redistribution, cytoplasm shrinkage, and nuclear deformation, the hallmarks of apoptosis. The observed tumor cell-specific addiction to the 26S proteasome levels sets the stage for future strategies in exploiting this dependency in cancer therapy.

Pax6 is essential for the generation of late-born retinal neurons and for inhibition of photoreceptor-fate during late stages of retinogenesis.

In the developing retina, as in other regions of the CNS, neural progenitors give rise to individual cell types during discrete temporal windows. Pax6 is expressed in retinal progenitor cells (RPCs) throughout the course of retinogenesis, and has been shown to be required during early retinogenesis for generation of most early-born cell types. In this study, we examined the function of Pax6 in postnatal mouse retinal development. We found that Pax6 is essential for the generation of late-born interneurons, while inhibiting photoreceptor differentiation. Generation of bipolar interneurons requires Pax6 expression in RPCs, while Pax6 is required for the generation of glycinergic, but not for GABAergic or non-GABAergic-non-glycinergic (nGnG) amacrine cell subtypes. In contrast, overexpression of either full-length Pax6 or its 5a isoform in RPCs induces formation of cells with nGnG amacrine features, and suppresses generation of other inner retinal cell types. Moreover, overexpression of both Pax6 variants prevents photoreceptor differentiation, most likely by inhibiting Crx expression. Taken together, these data show that Pax6 acts in RPCs to control differentiation of multiple late-born neuronal cell types.