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Nico P Dantuma

Publications and source records attributed to Nico P Dantuma.

At least 19 recordsLinked to original sources
PubMed2006

Ubiquitin crosstalk connecting cellular processes.

The polypeptide ubiquitin is used in many processes as different as endocytosis, multivesicular body formation, and regulation of gene transcription. Conjugation of a single ubiquitin moiety is typically used in these processes. A polymer of ubiquitin moieties is required for tagging proteins for proteasomal degradation. Besides its role in protein degradation, ubiquitin is also engaged as mono- or polymer in intracellular signalling and DNA repair. Since free ubiquitin is present in limiting amounts in cells, changes in the demands for ubiquitin in any of these processes is likely to indirectly affect other ubiquitin modifications. For example, proteotoxic stress strongly increases poly-ubiquitylated proteins at the cost of mono-ubiquitylated histones resulting in chromatin remodelling and altered transcription. Here we discuss the interconnection between ubiquitin-dependent processes and speculate on the functional significance of the ubiquitin equilibrium as a signalling route translating cellular stress into molecular responses.

Journal Article↗
PubMed2006

COPI-mediated retrograde transport is required for efficient gamma-secretase cleavage of the amyloid precursor protein.

Sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases results in the production of beta-amyloid peptide, which is a key determinant in Alzheimer's disease. Since several putative locations for gamma-secretase cleavage have been identified along the secretory pathway, trafficking of APP may be of importance for beta-amyloid peptide production. Here we have studied the role of retrograde transport in APP processing. We found that APP interacts with the beta subunit of the coatomer protein I (COPI) complex, which is involved in retrograde transport. In line with a role of retrograde trafficking in APP transport, inhibition of COPI-dependent transport altered APP trafficking, decreased APP cell surface expression, and coincided with a profound reduction in gamma-secretase cleavage. These results suggest that COPI-dependent retrograde transport is important for APP processing and influences production of beta-amyloid peptide.

Amyloid Precursor Protein Secretases↗
PubMed2006

DNA damage triggers nucleotide excision repair-dependent monoubiquitylation of histone H2A.

Chromatin changes within the context of DNA repair remain largely obscure. Here we show that DNA damage induces monoubiquitylation of histone H2A in the vicinity of DNA lesions. Ultraviolet (UV)-induced monoubiquitylation of H2A is dependent on functional nucleotide excision repair and occurs after incision of the damaged strand. The ubiquitin ligase Ring2 is required for the DNA damage-induced H2A ubiquitylation. UV-induced ubiquitylation of H2A is dependent on the DNA damage signaling kinase ATR (ATM- and Rad3-related) but not the related kinase ATM (ataxia telangiectasia-mutated). Although the response coincides with phosphorylation of variant histone H2AX, H2AX was not required for H2A ubiquitylation. Together our data show that monoubiquitylation of H2A forms part of the cellular response to UV damage and suggest a role of this modification in DNA repair-induced chromatin remodeling.

Amino Acid Sequence↗
PubMed2006

A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling.

Protein degradation, chromatin remodeling, and membrane trafficking are critically regulated by ubiquitylation. The presence of several coexisting ubiquitin-dependent processes, each of crucial importance to the cell, is remarkable. This brings up questions on how the usage of this versatile regulator is negotiated between the different cellular processes. During proteotoxic stress, the accumulation of ubiquitylated substrates coincides with the depletion of ubiquitylated histone H2A and chromatin remodeling. We show that this redistribution of ubiquitin during proteotoxic stress is a direct consequence of competition for the limited pool of free ubiquitin. Thus, the ubiquitin cycle couples various ubiquitin-dependent processes because of a rate-limiting pool of free ubiquitin. We propose that this ubiquitin equilibrium may allow cells to sense proteotoxic stress in a genome-wide fashion.

Active Transport, Cell Nucleus↗
PubMed2006

Non-infectious fluorimetric assay for phenotyping of drug-resistant HIV proteinase mutants.

BACKGROUND: The introduction of HIV proteinase inhibitors (PIs) as anti-AIDS drugs resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, rapid selection of drug-resistant HIV variants is a common complication in patients undergoing highly active anti-retroviral therapy (HAART). Thus, monitoring of clinical resistance development is indispensable for rational pharmacotherapy. OBJECTIVE: We present a non-infectious cell-based assay for drug resistance quantification of HIV proteinase (PR) - an important target of HAART. STUDY DESIGN: Previously, we showed [Lindsten K, Uhlikova T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother 2001;45:2616-22] that the expression of a fusion protein (GFP-PR), comprised of HIV-1 proteinase wild-type artificial precursor (PR) and green fluorescent protein (GFP), in transiently transfected tissue culture cells depends on the presence of PR-specific inhibitors (PIs). Here we show that in the GFP-PR reporter the HIV wild-type PR can be replaced by a drug-resistant HIV PR mutant, yielding a simple and biologically relevant tool for the quantitative analysis of drug-resistant HIV PR mutants susceptibility to HIV proteinase inhibitors. RESULTS: We cloned a set of GFP-PR reporters, some of which possess a simple, well-defined drug-resistant PR mutant (G48V L90M, V82A, A71V V82T I84V, D30N, K45I); another four complex PR mutants were obtained from patients undergoing HAART. The results were compared with genotyping and enzyme kinetics data. Furthermore, we designed a single inhibitor concentration experiment setup for easy evaluation of drug resistance profiles for mutants of interest. The resistance profiles clearly demonstrate the importance of succession of individual drugs during the treatment for drug resistance development. CONCLUSION: We show that the GFP-PR assay might serve as a non-infectious, rapid, cheap, and reliable alternative to the currently used phenotypic assays.

Antibodies, Monoclonal↗
PubMed2006

A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo.

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.

Animals↗
PubMed2005

Endoplasmic reticulum stress compromises the ubiquitin-proteasome system.

The presence of endoplasmic reticulum (ER) stress and impaired ubiquitin-proteasome system (UPS) activity has been independently implicated in the pathophysiology of conformational diseases. Here, we reveal a link between ER stress and the functionality of the UPS. Treatment of cells with different ER stressors delayed the degradation of an ER reporter substrate and caused a subtle but consistent accumulation of three independent nuclear/cytosolic UPS reporter substrates. A similar signature increase was observed upon induction of ER stress in transgenic mice expressing a reporter substrate. Cells undergoing ER stress failed to clear efficiently UBB+1, an aberrant ubiquitin found in conformational diseases, which in turn caused general impairment of the UPS. We conclude that ER stress has a general inhibitory effect on the UPS. The compromised UPS during ER stress may explain the long-term gradual accumulation of misfolded proteins as well as the selective vulnerability of particular cell populations in conformational diseases.

Animals↗
PubMed2005

The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation.

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.

Blotting, Western↗
PubMed2005

Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation.

The accumulation of protein deposits in neurons, in vitro proteasome assays and over-expression studies suggest that impairment of the ubiquitin-proteasome system (UPS) may be a common mechanism of pathogenesis in polyglutamine diseases such as Huntington disease and spinocerebellar ataxias (SCAs). Using a knock-in mouse model that recapitulates the clinical features of human SCA7, including selective neuronal dysfunction, we assessed the UPS at cellular resolution using transgenic mice that express a green fluorescent protein (GFP)-based reporter substrate (Ub(G76V)-GFP) of the UPS. The levels of the reporter remained low during the initial phase of disease, suggesting that neuronal dysfunction occurs in the presence of a functional UPS. Late in disease, we observed a significant increase in reporter levels specific to the most vulnerable neurons. Surprisingly, the basis for the increase in Ub(G76V)-GFP protein can be explained by a corresponding increase in Ub(G76V)-GFP mRNA in the vulnerable neurons. An in vitro assay also showed normal proteasome proteolytic activity in the vulnerable neurons. Thus, no evidence for general UPS impairment or reduction of proteasome activity was seen. The differential increase of Ub(G76V)-GFP among individual neurons directly correlated with the down-regulation of a marker of selective pathology and neuronal dysfunction in SCA7. Furthermore, we observed a striking inverse correlation between the neuropathology revealed by this reporter and ataxin-7 nuclear inclusions in the vulnerable neurons. Altogether, these data show a protective role against neuronal dysfunction for polyglutamine nuclear inclusions and exclude significant impairment of the UPS as a necessary step for polyglutamine neuropathology.

Animals↗
PubMed2005

Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates.

A reliable and robust means of evaluating the functional status of ubiquitin-dependent proteolysis in living cells is to follow the turnover of readily detectable reporter substrates. During the past few years, several reporter substrates have been generated by use of the green fluorescent protein (GFP), which is converted for this purpose from a normally very stable protein into a short-lived substrate of the ubiquitin/proteasome system. These short-lived substrates are valuable tools providing researchers with unique information about the absence or presence of blockades in this system in living cells. We have recently generated the first transgenic mouse model for monitoring the ubiquitin/proteasome system based on the ubiquitous expression of a GFP-based proteasome substrate. Together these models can be used to study ubiquitin-dependent degradation in health and disease and for the identification of small synthetic compounds or proteins capable of modifying the activity of the system. In this chapter, we describe the basic principles of GFP-based reporter substrates, their strengths and weaknesses, and a number of protocols that can be used to study the ubiquitin/proteasome system in yeast, cell lines, and transgenic mice.

Animals↗
PubMed2005

Fluorescent reporters for the ubiquitin-proteasome system.

Regulated turnover of proteins in the cytosol and nucleus of eukaryotic cells is primarily performed by the ubiquitin-proteasome system (UPS). The UPS is involved in many essential cellular processes. Alterations in this proteolytic system are associated with a variety of human pathologies, such as neurodegenerative diseases, cancer, immunological disorders and inflammation. The precise role of the UPS in the pathophysiology of these diseases, however, remains poorly understood. Detection of UPS aberrations has been a major challenge because of the complexity of the system. Most studies focus on various aspects of the UPS, such as substrate recognition, ubiquitination, deubiquitination or proteasome activity, and do not provide a complete picture of the UPS as an integral system. To monitor the efficacy of the UPS, a number of reporter substrates have been developed based on fluorescent proteins, such as the green fluorescent protein and its spectral variants. These fluorescent UPS reporters contain specific degradation signals that target them with high efficiency and accuracy for proteasomal degradation. Several studies have shown that these reporters can probe the functionality of the UPS in cellular and animal models and provide us with important information on the status of the UPS under various conditions. Moreover, these reporters can aid the identification and development of novel anti-cancer and anti-inflammatory drugs based on UPS inhibition.

Animals↗
PubMed2003

Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat.

The glycine-alanine (GA) repeat of the Epstein-Barr virus nuclear antigen-1 inhibits in cis ubiquitin-dependent proteolysis in mammalian cells through a yet unknown mechanism. In the present study we demonstrate that the GA repeat targets an evolutionarily conserved step in proteolysis since it can prevent the degradation of proteasomal substrates in the yeast Saccharomyces cerevisiae. Insertion of yeast codon-optimised recombinant GA (rGA) repeats of different length in green fluorescent protein reporters harbouring N-end rule or ubiquitin fusion degradation signals resulted in efficient stabilisation of these substrates. Protection was also achieved in rpn10delta yeast suggesting that this polyubiquitin binding protein is not required for the rGA effect. The conserved effect of the GA repeat in yeast opens the possibility for the use of genetic screens to unravel its mode of action.

Alanine↗
PubMed2003

A transgenic mouse model of the ubiquitin/proteasome system.

Impairment of the ubiquitin/proteasome system has been proposed to play a role in neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although recent studies confirmed that some disease-related proteins block proteasomal degradation, and despite the existence of excellent animal models of both diseases, in vivo data about the system are lacking. We have developed a model for in vivo analysis of the ubiquitin/proteasome system by generating mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal. Administration of proteasome inhibitors to the transgenic animals resulted in a substantial accumulation of GFP in multiple tissues, confirming the in vivo functionality of the reporter. Moreover, accumulation of the reporter was induced in primary neurons by UBB+1, an aberrant ubiquitin found in Alzheimer disease. These transgenic animals provide a tool for monitoring the status of the ubiquitin/proteasome system in physiologic or pathologic conditions.

Alzheimer Disease↗
PubMed2003

The ubiquitin/proteasome system in Epstein-Barr virus latency and associated malignancies.

Epstein-Barr virus (EBV) is a human herpesvirus associated with lymphoid and epithelial malignancies. Three viral proteins, the EBV nuclear antigen 1 and the latent membrane proteins-1 and -2A, regulate viral latency by manipulating ubiquitin-dependent proteolysis. The activation of cellular oncogenes is instrumental for the progression of EBV infected cells to full malignancy. Constitutively activation of c-myc correlates with decreased proteasomal activity and upregulation of compensatory proteolytic pathways in Burkitt's lymphomas. Knowledge of these multiple strategies of interference with regulated proteolysis may provide new clues for the treatment of EBV-associated malignancies.

Burkitt Lymphoma↗
PubMed2003

Monitoring the ubiquitin/proteasome system in conformational diseases.

Controlled proteolysis of regulatory or aberrant proteins by the ubiquitin/proteasome system is indispensable for cell viability. Conformational diseases such as Alzheimer's, Parkinson's and Huntington's disease are characterised by the accumulation of misfolded or aggregation-prone proteins. Since these proteins are typical substrates of the ubiquitin/proteasome system, it is not surprising that various models propose impairment of this system as a contributing factor to the pathology of conformational disorders. The complex nature of the ubiquitin/proteasome system and its universal role in cell physiology however turns evaluation of these attractive hypotheses into a major challenge. Several reporter substrates for the ubiquitin/proteasome system have recently been developed to facilitate functional studies of the system in living cells. In this review, we will discuss these new tools as well as the proteins associated with conformational disease that have been studied with these reporters.

Animals↗
PubMed2002

Pathways accessory to proteasomal proteolysis are less efficient in major histocompatibility complex class I antigen production.

Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal beta-subunits (beta(1), beta(2), and beta(5)) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2D(b) molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways.

Animals↗