Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MITcontaining proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.Lysosomal degradation rates determine the levels of cell surface receptor tyrosine kinases, an important parameter in the control of cell growth (1, 2). Activated receptors are internalized and consequently committed to the lysosomal pathway by budding from the limiting membrane of the early endosome into lumenal vesicles, which define the multivesicular body (MVB).3 Ubiquitination of receptors is required for their sorting into MVBs, which precludes their recycling to the plasma membrane (3,4). The constituents of the endosomal sorting machinery were initially identified as class E mutants in screens for vacuolar protein-sorting (VPS) defects in Saccharomyces cerevisiae (5,6), characterized by an expanded pre-vacuolar compartment (7). These engage in a complex set of protein-protein interactions, which link four core complexes (endosomal sorting complexes required for transport, ESCRTs-0, I, II and III), and somehow impart directionality to the process (5, 8 -11). It has been proposed that ESCRT-0 (comprising Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule)) may provide the first means of engagement with ubiquitinated receptor (12-15) through ubiquitin interaction motifs in both proteins, although both ESCRT-I and -II also contain ubiquitin-binding proteins (10,16). In yeast, ESCRT-III is composed of four subunits (VPS2/Chm2, VPS24/Chm3, Snf7/Chm4, VPS20/Chm6), whereas mammals possess an expanded complement of isoforms, CHMP2A/B, CHMP3, CHMP4A/B/C, and CHMP6, providing the possibility for distinct ESCRT-III functions through combinatorial coding of core components. Two related proteins, Did2/VPS46/Chm1 and VPS60/MOS10/Chm5 (CHMP1A/B and CHMP5 in mammals), have poorly defined auxiliary roles in MVB sorting (17)(18)(19), and a further mammalian CHMP protein (CHMP7) has no yeast orthologue (20). All the CHMPs...

The endosomal pathway provides a major platform for ubiquitin-modifying enzymes, which act upon membrane-associated proteins in transit. Ubiquitylated cargo proteins are recognized by ubiquitin-binding domains inherent to key adaptor proteins at the plasma membrane and sorting endosome. A balance between ubiquitylation and deubiquitylation activities may govern the efficiency of recycling from endosomes to the plasma membrane versus lysosomal sorting through the multivesicular body pathway. We discuss the current knowledge of the properties of adaptors and ubiquitin-modifying proteins and their effects upon the trafficking and signaling of receptors and ligands associated with pathways fundamental to development.

A localization atlas is provided for 66 of 90 mammalian GFP-tagged deubiquitinases (DUBs). USP21 is the only DUB in the panel that localizes to both microtubules and the centrosome. Functional data suggest a key role for USP21 in the choreography of microtubule reorganization.

Based on their electronic conductivity behaviour, metallic nanowires may have analytical applications ranging from interconnects to sensors. We present in this paper an electrochemical method for synthesizing Mo and Pd metal nanowires ranging in diameter from a few tens of nanometres up to one micrometre, with millimetre lengths. Nanowires are prepared by the electrodeposition of metal at step edges present on a graphite surface. These nanowires can be used to connect metal nanoparticles (Ni, Au, etc.). Once transferred in a polymer cast, they can operate as sensors. We describe how these nanowires may be manipulated to make devices for analytical chemistry and, as an example of such nanodevice, a detailed overview of the characteristics of the first nanowire-based sensor of hydrogen gas (H 2 ) is given. Copyright  2002 John Wiley & Sons, Ltd. KEYWORDS: nanowire; nanoparticle; electrodeposition; sensor; palladium; hydrogen Chemical sensing is about surface and interface interactions between the analyte molecules and the sensing material. In that sense, nano-objects with a large surface atombulk atom ratio, such as nanoparticles and nanowires, are potentially very efficient chemical sensors. The mechanism envisaged involves adsorption (and eventually diffusion) of the analyte molecule at the surface, which induces a change in the electrical resistance of the nano-object: sensing is the process of measuring these conductivity changes. The most convenient way to measure conductivity changes in such devices is to obtain the specific material as nanowires or as connected nanoparticles. Although chemically selective sensors operating on this principle do not yet exist, Tao and coworkers have shown changes in the conductivity of gold nanowires upon exposure to thiols or amines. 1In the same way, single-walled carbon nanotubes exhibit resistance changes upon exposure to gaseous oxygen, water and amines. 2 We describe in this paper an electrochemical method for the preparation of naked (palladium) and bimetallic beaded nanowires in which nickel, gold or palladium beads are connected by molybdenum or palladium nanowires. We also show how to manipulate these objects to build chemical sensors. As an example, the characteristics of a nanowirebased sensor for the detection of hydrogen gas are presented. EXPERIMENTALSensors described hereafter consist of up to 100 nanowires arrayed in parallel, as shown in Fig. 1(a). These arrays were prepared by electrodeposition at step edges present Ł Correspondence to: F. Favier, UMR 5072 CNRS, Université Montpellier II, cc015, 34095 Montpellier Cedex 05, France. E-mail: fredf@univ-montp2.fr on a graphite surface, as shown schematically in Fig. 1(b). For beaded nanowires, nickel, gold and palladium particles were first electrodeposited from aqueous solutions of Ni 2C , Au 3C or Pd 2C respectively. In a second step, nanowires of molybdenum oxide (MoO x ) or palladium were grown from MoO 4 2 or Pd 2C solutions, respectively. Both particles and wires were prepared according to methods descri...

ESCRT-0 component HRS and actin polymerization factor WASH reside in adjacent endosomal domains. MacDonald et al. show that HRS controls WASH localization and recycling of WASH-dependent transmembrane cargo. Cargo binding to endosomal actin thus acts as sorting signal to oppose ubiquitin-mediated degradation.

Morphogenesis in fungi is often induced by extracellular factors and executed by fungal genetic factors. Cell surface changes and alterations of the microenvironment often accompany morphogenetic changes in fungi. In this review, we will first discuss the general traits of yeast and hyphal morphotypes and how morphogenesis affects development and adaptation by fungi to their native niches, including host niches. Then we will focus on the molecular machinery responsible for the two most fundamental growth forms, yeast and hyphae. Last, we will describe how fungi incorporate exogenous environmental and host signals together with genetic factors to determine their morphotype and how morphogenesis, in turn, shapes the fungal microenvironment. PROPERTIES OF MORPHOGENESIS IN FUNGAL CELLS

The phosphatidylinositol-3-kinase (PI3K) pathway is commonly hyperactivated in cancer. One mechanism by which this occurs is by silencing of the phosphatase and tensin homolog (PTEN), a tumor suppressor and major antagonist of the pathway, through genetic, epigenetic or posttranscriptional mechanisms. Here, we used an unbiased siRNA screen in non-small-cell lung cancer cells to identify deubiquitylases (DUBs) that have an impact on PI3K signaling by regulating the abundance of PTEN. We found that PTEN expression was induced by depleting any of three members of the Josephin family DUBs: ataxin 3 (ATXN3), ataxin 3-like (ATXN3L) and Josephin domain containing 1 (JOSD1). However, this effect is not mediated through altered PTEN protein stability. Instead, depletion of each DUB increases expression of both the PTEN transcript and its competing endogenous RNA, PTENP1. In ATXN3-depleted cells, under conditions of transcriptional inhibition, PTEN and PTENP1 mRNAs rapidly decay, suggesting that ATXN3 acts primarily by repressing their transcription. Importantly, the PTEN induction observed in response to ATXN3 siRNA is sufficient to downregulate Akt phosphorylation and hence PI3K signaling. Histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in non-small-cell lung cancer. Although PTEN exhibits a very limited response to the broad-spectrum HDACi Vorinostat (SAHA) in A549 cells, we find that combination with ATXN3 depletion enhances PTEN induction in an additive manner. Similarly, these interventions additively decrease cell viability. Thus, ATXN3 provides an autonomous, complementary therapeutic target in cancers with epigenetic downregulation of PTEN.