Biomedical subjects

Simona Polo

Publications and source records attributed to Simona Polo.

15 recordsLinked to original sources

PubMed2006

Endocytosis conducts the cell signaling orchestra.

Endocytosis is used by eukaryotic cells to regulate nutrient internalization, signal transduction, and the composition of the plasma membrane. However, a more complex picture is emerging, in which endocytic pathways integrate diverse signals, thereby contributing to a higher level of cellular and organismal organization. In this way, endocytosis and cell signaling are intertwined in many biological processes, such as cell motility and cell fate determination.

Cell Differentiation↗

PubMed2006

Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin.

The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.

Amino Acid Sequence↗

PubMed2005

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes.

Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.

Actins↗

PubMed2005

Deubiquitinating function of ataxin-3: insights from the solution structure of the Josephin domain.

Spinocerebellar ataxia type 3 is a human neurodegenerative disease resulting from polyglutamine tract expansion. The affected protein, ataxin-3, which contains an N-terminal Josephin domain followed by tandem ubiquitin (Ub)-interacting motifs (UIMs) and a polyglutamine stretch, has been implicated in the function of the Ub proteasome system. NMR-based structural analysis has now revealed that the Josephin domain binds Ub and has a papain-like fold that is reminiscent of that of other deubiquitinases, despite primary sequence divergence but consistent with its deubiqutinating activity. Mutation of the catalytic Cys enhances the stability of a complex between ataxin-3 and polyubiquitinated proteins. This effect depends on the integrity of the UIM region, suggesting that the UIMs are bound to the substrate polyubiquitin during catalysis. We propose that ataxin-3 functions as a polyubiquitin chain-editing enzyme.

Ataxin-3↗

PubMed2005

N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits.

WASP and WAVE family proteins promote actin polymerization by stimulating Arp2/3-complex-dependent filament nucleation. Unlike WAVE proteins, which are known to drive the formation of protrusions such as lamellipodia and membrane ruffles, vertebrate cell functions of WASP or N-WASP are less well established. Recent work demonstrated that clathrin-coated pit invagination can coincide with assembly of actin filaments and with accumulation of N-WASP and Arp2/3 complex, but the relevance of their recruitment has remained poorly defined. We employed two-colour total internal reflection microscopy to study the recruitment and dynamics of various components of the actin polymerization machinery and the epidermal growth factor receptor signalling machinery during clathrin-coated pit internalization in control cells and cells genetically deficient for functional N-WASP. We found that clathrin-coated pit endocytosis coincides with the recruitment of N-WASP, Arp2/3 complex and associated proteins, but not of WAVE family members. Actin accumulation at clathrin-coated pits requires the Arp2/3 complex, since Arp2/3 complex sequestration in the cytosol abolished any detectable actin assembly. The absence of N-WASP caused a significant reduction in the frequencies of actin and Arp2/3 complex accumulations at sites of clathrin-coated pit invagination and vesicle departure. Although N-WASP was not essential for Arp2/3-complex-mediated actin assembly at these sites or for EGF receptor-mediated endocytosis, N-WASP deficiency caused a marked reduction of EGF internalization. We conclude that the assembly of WASP subfamily proteins and associated factors at sites of clathrin-coated pit invagination amplifies actin accumulations at these sites promoting efficient internalization of ligands via clathrin-mediated endocytosis.

Actin-Related Protein 2-3 Complex↗

PubMed2005

Clathrin-independent endocytosis of ubiquitinated cargos.

Plasma membrane receptors can be endocytosed through clathrin-dependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR/ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Endocytosis and cancer.

Eukaryotic cells use endocytosis to internalise plasma membrane, surface receptors and their ligands, viruses and various extracellular soluble molecules. Endocytosis has been regarded as a long-term mechanism of signal attenuation via receptor clearance from the cell surface. However, additional, and quite unexpected, functions for endocytosis have emerged, which, together with its attenuation function, project a central role for this process in cellular homeostasis and control of proliferation. Subversion of endocytic control is thus predicted to play a causative role in hyperproliferative conditions, first and foremost cancer.

Animals↗

PubMed2003

EH and UIM: endocytosis and more.

Exogenously and endogenously originated signals are propagated within the cell by functional and physical networks of proteins, leading to numerous biological outcomes. Many protein-protein interactions take place between binding domains and short peptide motifs. Frequently, these interactions are inducible by upstream signaling events, in which case one of the two binding surfaces may be created by a posttranslational modification. Here, we discuss two protein networks. One, the EH-network, is based on the Eps15 homology (EH) domain, which binds to peptides containing the sequence Asp-Pro-Phe (NPF). The other, which we define as the monoubiquitin (mUb) network, relies on monoubiquitination, which is emerging as an important posttranslational modification that regulates protein function. Both networks were initially implicated in the control of plasma membrane receptor endocytosis and in the regulation of intracellular trafficking routes. The ramifications of these two networks, however, appear to extend into many other aspects of cell physiology as well, such as transcriptional regulation, actin cytoskeleton remodeling, and DNA repair. The focus of this review is to integrate available knowledge of the EH- and mUb networks with predictions of genetic and physical interactions stemming from functional genomics approaches.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Rapid Ca2+-dependent decrease of protein ubiquitination at synapses.

Protein ubiquitination has been implicated in the regulation of axonal growth and synaptic plasticity as well as in the pathogenesis of neurodegenerative diseases. Here we show that depolarization-dependent Ca2+ influx into synaptosomes produces a global, rapid (range of seconds), and reversible decrease of the ubiquitinated state of proteins, which correlates with the Ca2+-dependent dephosphorylation of several synaptic proteins. A similar general decrease in protein ubiquitination was observed in nonneuronal cells on Ca2+ entry induced by ionomycin. Both in synaptosomes and in nonneuronal cells, this decrease was blocked by FK506 (a calcineurin antagonist). Proteins whose ubiquitinated state was decreased include epsin 1, a substrate for the deubiquitinating enzyme fat facets/FAM, which we show here to be concentrated at synapses. These results reveal a fast regulated turnover of protein ubiquitination. In nerve terminals, protein ubiquitination may play a role both in the regulation of synaptic function, including vesicle traffic, and in the coordination of protein turnover with synaptic use.

Animals↗

PubMed2003

Characterization of human constitutive photomorphogenesis protein 1, a RING finger ubiquitin ligase that interacts with Jun transcription factors and modulates their transcriptional activity.

RING finger proteins have been implicated in many fundamental cellular processes, including the control of gene expression. A key regulator of light-dependent development in Arabidopsis thaliana is the constitutive photomorphogenesis protein 1 (atCOP1), a RING finger protein that plays an essential role in translating light/dark signals into specific changes in gene transcription. atCOP1 binds the basic leucine zipper factor HY5 and suppresses its transcriptional activity through a yet undefined mechanism that results in HY5 degradation in response to darkness. Furthermore, the pleiotropic phenotype of atCOP1 mutants indicates that atCOP1 may be a central regulator of several transcriptional pathways. Here we report the cloning and characterization of the human orthologue of atCOP1. Human COP1 (huCOP1) distributes both to the cytoplasm and the nucleus of cells and shows a striking degree of sequence conservation with atCOP1, suggesting the possibility of a functional conservation as well. In co-immunoprecipitation assays huCOP1 specifically binds basic leucine zipper factors of the Jun family. As a functional consequence of this interaction, expression of huCOP1 in mammalian cells down-regulates c-Jun-dependent transcription and the expression of the AP-1 target genes, urokinase and matrix metalloproteinase 1. The RING domain of huCOP1 displays ubiquitin ligase activity in an autoubiquitination assay in vitro; however, suppression of AP-1-dependent transcription by huCOP1 occurs in the absence of changes in c-Jun protein levels, suggesting that this inhibitory effect is independent of c-Jun degradation. Our findings indicate that huCOP1 is a novel regulator of AP-1-dependent transcription sharing the important properties of Arabidopsis COP1 in the control of gene expression.

Amino Acid Sequence↗

PubMed2003

Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation.

Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated - a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.

Animals↗

PubMed2003

When ubiquitin meets ubiquitin receptors: a signalling connection.

Ubiquitylation is emerging as a versatile device for controlling cellular functions. Here, we propose that monoubiquitylation is rapidly induced by signalling events and allows the establishment of protein-protein interactions between monoubiquitylated proteins and partners that contain distinct ubiquitin-binding domains. We also put forward speculative models for the regulation of monoubiquitylation versus polyubiquitylation.

Animals↗

PubMed2002

A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins.

Ubiquitination is a post-translation modification in which ubiquitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or monoubiquitination, respectively. Polyubiquitination targets proteins for destruction by the proteasome. The role of monoubiquitination is less understood, although a function in membrane trafficking is emerging, at least in yeast. Here we report that a short amino-acid stretch at the carboxy-termini of the monoubiquitinated endocytic proteins Eps15 and eps15R is indispensable for their monoubiquitination. A similar sequence, also required for this modification, is found in other cytosolic endocytic proteins, such as epsins and Hrs. These sequences comprise a protein motif, UIM (ref. 6), which has been proposed to bind to ubiquitin. We confirm this for the UIMs of eps15, eps15R, epsins and Hrs. Thus, the same motif in several endocytic proteins is responsible for ubiquitin recognition and monoubiquitination. Our results predict the existence of a UIM:ubiquitin-based intracellular network. Eps15/eps15R, epsins and Hrs may function as adaptors between ubiquitinated membrane cargo and either the clathrin coat or other endocytic scaffolds. In addition, through their own ubiquitination, they may further contribute to the amplification of this network in the endocytic pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Differential nucleocytoplasmic trafficking between the related endocytic proteins Eps15 and Eps15R.

Eps15 and Eps15R are constitutive components of clathrin-coated pits that are required for clathrin-dependent endocytosis. The most striking difference between these two related proteins is that Eps15R is also found in the nucleus, whereas Eps15 is excluded from this compartment at steady state. To better understand the individual functions of these two proteins, the mechanisms responsible for their different localization were investigated. Interestingly, some mutants of Eps15 were found in the nucleus. This nuclear localization was correlated with the loss of the last approximately 100 amino acids of Eps15, suggesting the presence of a nuclear export signal (NES) within this region. As expected, the last 25 amino acids contain a leucine-rich sequence matching with classical NESs, show a leptomycin B-sensitive nuclear export activity, and bind to the exportin CRM1 in a leucine residue-dependent manner. In contrast, no NES could be found in Eps15R, a result in keeping with its constitutive nuclear localization that appears to be regulated by alternative splicing. Altogether, these results are the first characterization of nucleocytoplasmic shuttling signals for endocytic proteins. They also provide an explanation for the different nuclear localization of Eps15 and Eps15R and further evidence for a possible nuclear function for Eps15 protein family members.

3T3 Cells↗

PubMed1998

Identification of two linear plasmids in the actinomycete Planobispora rosea.

Two linear plasmids (pPR1, 27.5 kb, and pPR2, 16 kb) were identified in Planobispora rosea, an actinomycete that produces the antibiotic GE2270, an inhibitor of the elongation factor Tu. Strains lacking both plasmids still produce and are resistant to GE2270. The two plasmids share an internal region of high similarity, but no cross-hybridization was detected between their telomeric regions or between plasmid and chromosomal DNA. The 5' ends of the plasmids appear to be linked to terminal proteins. The telomeric regions of pPR2 were cloned after 3'-end homopolymer tailing and PCR amplification. The approximately 650 nt telomeric DNA sequences of pPR2 are repeated in inverted orientation and are rich in direct and inverted repeats; the 350 bp terminal region is less G + C-rich than the rest of the plasmid. The structural organization of these plasmids appears to be similar to Streptomyces linear replicons.

Actinomycetales↗