Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway

The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10delta) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimdelta)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10delta cells, whereas only one-fifth as many accumulated in uimdelta cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS.     

The molecular basis of the plumage colour polymorphism in the Tahiti reed‐warbler Acrocephalus caffer

The endemic Tahiti reed‐warbler Acrocephalus caffer occurs in two distinct morphs, a typical or ‘yellow’ morph and a melanic or ‘dark’ morph, which are found together in the valleys of the eastern and central parts of the island of Tahiti (Society Islands, French Polynesia). We investigated the molecular basis of the plumage colour polymorphism in this species using sequences of the melanocortin‐1 receptor (MC1R), a gene often found associated to melanism in birds. We found that the MC1R genotype was perfectly associated with plumage colour in the Tahiti reed‐warbler, with the same nonsynonymous substitution that showed a correlation with phenotype in the Caribbean bananaquit Coereba flaveola. An heterozygous reed‐warbler at this site presented a melanic phenotype, suggesting that the melanic allele is dominant. All other Polynesian reed‐warbler species, which do not have a melanic morph, shared the ‘yellow’ nucleotide at this position. These results suggested that the same mutation point was linked to a melanic polymorphism in two unrelated passerine birds.     

Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone

The ClpXP ATPase-protease complex is a major component of the protein quality control machinery in the cell. A ClpX subunit consists of an N-terminal zinc binding domain (ZBD) and a C-terminal AAA+ domain. ClpX oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the ZBDs extending out of the base. Here, we report that ClpX switches between a capture and a feeding conformation. ZBDs in ClpX undergo large nucleotide-dependent block movement towards ClpP and into the AAA+ ring. This motion is modulated by the ClpX cofactor, SspB. Evidence for this movement was initially obtained by the surprising observation that an N-terminal extension on ClpX is clipped by bound ClpP in functional ClpXP complexes. Protease-protection, crosslinking, and light scattering experiments further support these findings.     

Time-resolved circular dichroism in carbonmonoxy-myoglobin: the central role of the proximal histidine

A calculation of the circular dichroism (CD) spectra of carbonmonoxy- and deoxy-myoglobin is carried out in relation to a time-resolved CD experiment. This calculation allows us to assign a dominant role to the proximal histidine in the definition of the electronic normal modes and to interpret the transient CD structure observed in a strain of the proximal histidine. This strain builds up in 10 ps and relaxes in 50 ps as the protein evolves towards its deoxy form.     

Migration of bone marrow stromal cells in 3D: 4 color methodology reveals spatially and temporally coordinated events

The cytoskeleton plays a central role in many cell processes including directed cell migration. Since most previous work has investigated cell migration in two dimensions (2D), new methods are required to study movement in three dimensions (3D) while preserving 3D structure of the cytoskeleton. Most previous studies have labeled two cytoskeletal networks simultaneously, impeding an appreciation of their complex and dynamic interconnections. Here we report the development of a 4 color method to simultaneously image vimentin, actin, tubulin and the nucleus for high-resolution confocal microscopy of bone-marrow stromal cells (BMSCs) migrating through a porous membrane. Several methods were tested for structural preservation and labeling intensity resulting in identification of an optimized simultaneous fixation and permeabilization method using glutaraldehyde, paraformaldehyde and Triton X-100 followed by a quadruple fluorescent labeling method. This procedure was then applied at a sequence of time points to migrating cells, allowing temporal progression of migration to be assessed by visualizing all three networks plus the nucleus, providing new insights into 3D directed cell migration including processes such as leading edge structure, cytoskeletal distribution and nucleokinesis. Colocalization of actin and microtubules with distinct spatial arrangements at the cellular leading edge during migration, together with microtubule axial polarization supports recent reports indicating the pivotal role of microtubules in directed cell migration. This study also provides a foundation for 3D migration studies versus 2D studies, providing precise and robust methods to attain new insights into the cellular mechanisms of motility.     

Improved anti-tumoral capacity of mixed and pure anti-oestrogens in breast cancer cell xenografts after their administration by entrapment in colloidal nanosystems

Anti-oestrogens (AEs) are currently used for treating hormone-dependent breast cancers. They specifically bind to oestrogen receptors (ERs) and inhibit their transactivation capacity. However, ERs are present in various other tissues in which AEs may have either a beneficial or detrimental action. AE administration via systems targeting breast tumours may be an important therapeutic improvement. Thus, several biodegradable drug delivery systems containing either “mixed” (4-hydroxytamoxifen - 4-HT) or “pure” (RU 58668 - RU) AEs were prepared. Liposomes and nanospheres (NS, composed of non-toxic and biodegradable lipids and poly(d,l-lactic acid) incorporated up to 1 and 0.5 mM AE, respectively. Nanocapsules (NCs) in which an oily core solubilises the AE incorporated no more than 0.02 mM of the drug. PEG-functionalised nanoparticles survived longer in plasma and had better controlled release of the drug. The small size of the vectors (100–250 nm) was compatible with their extravasation through the discontinuous endothelium of tumour vasculature, allowing their accumulation in MCF-7 cell xenografts and leading to a prolonged exposure of the tumour to AEs. In these tumours and in MCF-7/ras xenografts, RU-NS and RU-NC (6.5 mg/kg/week and 0.27 mg/kg/week, respectively, doses at which free RU had a very weak effect), both inhibited tumour growth. Entrapped RU significantly induced involution of tumours and strongly induced apoptosis in tumour cells, concomitantly with inhibiting tumour angiogenesis. 4-HT-nanoparticles also arrest oestradiol-induced tumour growth, inducing apoptosis and inhibiting angiogenesis. However, unlike RU-nanoparticles, they did not promote ER subtype loss in tumour cells. Subcutaneous administration of both RU- and 4-HT-NS in MCF-7 xenografts strongly arrested tumour growth for prolonged periods and RUNS decreased the number of tumour epithelial cells. Analysis of the proteins involved in cell cycle proliferation and apoptosis confirmed that RU-nanoparticles were more efficient than 4-HT-nanoparticles. Their lack of toxicity and high anti-tumour potency that affects only tumour cells in the xenograft models mean these AE-loaded colloidal systems are a breakthrough in hormone-dependent breast cancer treatment.     

Distinct modulation of voltage-gated and ligand-gated Ca2+ currents by PPAR-γ agonists in cultured hippocampal neurons

Type 2 diabetes mellitus is a metabolic disorder characterized by hyperglycemia and is especially prevalent in the elderly. Because aging is a risk factor for type 2 diabetes mellitus, and insulin resistance may contribute to the pathogenesis of Alzheimer's disease (AD), anti-diabetic agents (thiazolidinediones-TZDs) are being studied for the treatment of cognitive decline associated with AD. These agents normalize insulin sensitivity in the periphery and can improve cognition and verbal memory in AD patients. Based on evidence that Ca²⁺ dysregulation is a pathogenic factor of brain aging/AD, we tested the hypothesis that TZDs could impact Ca²⁺ signaling/homeostasis in neurons. We assessed the effects of pioglitazone and rosiglitazone (TZDs) on two major sources of Ca²⁺ influx in primary hippocampal cultured neurons, voltage-gated Ca²⁺ channel (VGCC) and the NMDA receptor (NMDAR). VGCC- and NMDAR-mediated Ca²⁺ currents were recorded using patch-clamp techniques, and Ca²⁺ intracellular levels were monitored with Ca²⁺ imaging techniques. Rosiglitazone, but not pioglitazone reduced VGCC currents. In contrast, NMDAR-mediated currents were significantly reduced by pioglitazone but not rosiglitazone. These results show that TZDs modulate Ca²⁺-dependent pathways in the brain and have different inhibitory profiles on two major Ca²⁺ sources, potentially conferring neuroprotection to an area of the brain that is particularly vulnerable to the effects of aging and/or AD.     

Spatio‐temporal control of neural epithelial cell migration and epithelium‐to‐mesenchyme transition during avian neural tube development

As opposed to the neural crest, the neural epithelium is generally viewed as a static and cohesive structure. Here, using an ex vivo system free of the environmental influences and physical constraints encountered in the embryo, we show that neural epithelial cells are on the contrary intrinsically motile, although they do not undergo spontaneous epithelium‐to‐mesenchyme transition and display molecular and cellular characteristics distinct from those of neural crest cells. However, they can be instructed to undergo epithelium‐to‐mesenchyme conversion independently of the acquisition of neural crest traits. Migration potentialities of neural epithelial cells are transient and are progressively restricted during neural tube development. Restriction of cell migration is irreversible and can be in part accounted for by increase in N‐cadherin in cellular junctions and in cell polarity. In conclusion, our study reveals that the neural epithelium is a highly flexible tissue in which cells are maintained cohesive under the control of a combination of extrinsic factors and physical constraints.