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Biomedical subjects

M Piechaczyk

Publications and source records attributed to M Piechaczyk.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a C-terminal tripeptide motif involved in the control of rapid proteasomal degradation of c-Fos proto-oncoprotein during the G(0)-to-S phase transition.

c-Fos proto-oncoprotein is rapidly and transiently expressed in cells undergoing the G(0)-to-S phase transition in response to stimulation for growth by serum. Under these conditions, the rapid decay of the protein occurring after induction is accounted for by efficient recognition and degradation by the proteasome. PEST motifs are sequences rich in Pro, Glu, Asp, Ser and Thr which have been proposed to constitute protein instability determinants. c-Fos contains three such motifs, one of which comprises the C-terminal 20 amino acids and has already been proposed to be the major determinant of c-Fos instability. Using site-directed mutagenesis and an expression system reproducing c-fos gene transient expression in transfected cells, we have analysed the turnover of c-Fos mutants deleted of the various PEST sequences in synchronized mouse embryo fibroblasts. Our data showed no role for the two internal PEST motifs in c-Fos instability. However, deletion of the C-terminal PEST region led to only a twofold stabilization of the protein. Taken together, these data indicate that c-Fos instability during the G0-to-S phase transition is governed by a major non-PEST destabilizer and a C-terminal degradation-accelerating element. Further dissection of c-Fos C-terminal region showed that the degradation-accelerating effect is not contributed by the whole PEST sequence but by a short PTL tripeptide which cannot be considered as a PEST motif and which can act in the absence of any PEST environment. Interestingly, the PTL motif is conserved in other members of the fos multigene family. Nevertheless, its contribution to protein instability is restricted to c-Fos suggesting that the mechanisms whereby the various Fos proteins are broken down are, at least partially, different. MAP kinases-mediated phosphorylation of two serines close to PTL, which are both phosphorylated all over the G(0)-to-S phase transition, have been proposed by others to stabilize c-Fos protein significantly. We, however, showed that the PTL motif does not exert its effect by counteracting a stabilizing effect of these phosphorylations under our experimental conditions.

3T3 Cells↗
PubMed2001

Cellular and viral Fos proteins are degraded by different proteolytic systems.

c-Fos proto-oncoprotein is a short-lived transcription factor degraded by the proteasome in vivo. Its mutated forms expressed by the mouse osteosarcomatogenic retroviruses, FBJ-MSV and FBR-MSV, are stabilized two- and threefold, respectively. To elucidate the mechanisms underlying v-Fos(FBJ) and v-Fos(FBR) protein stabilization, we conducted a genetic analysis in which the half-lives and the sensitivities to various cell-permeable protease inhibitors of a variety of cellular and viral protein mutants were measured. Our data showed that the decreased degradation of v-Fos(FBJ) and v-Fos(FBR) is not simply explained by the deletion of a c-Fos destabilizing C-terminal domain. Rather, it involves a complex balance between opposing destabilizing and stabilizing mutations which are distinct and which include virally-introduced peptide motifs in both cases. The mutations in viral Fos proteins conferred both total insensitivity to proteasomal degradation and sensitivity to another proteolytic system not naturally operating on c-Fos, explaining the limited stabilization of the two proteins. This observation is consistent with the idea that FBR-MSV and FBJ-MSV expression machineries have evolved to ensure controlled protein levels. Importantly, our data illustrate that the degradation of unstable proteins does not necessarily involve the proteasome and provide support to the notion that highly related proteins can be broken down by different proteolytic systems in living cells.

Amino Acid Sequence↗
PubMed2001

Human cyclin C protein is stabilized by its associated kinase cdk8, independently of its catalytic activity.

Cyclin C belongs to the cyclin family of proteins that control cell cycle transitions through activation of specific catalytic subunits, the cyclin-dependent kinases (CDKs). However, there is as yet no evidence for any role of cyclin C and its partner, cdk8, in cell cycle regulation. Rather, the cyclin C-cdk8 complex was found associated with the RNA polymerase II transcription machinery. The periodic degradation of bona fide cyclins is crucial for cell-cycle progression and depends on the catalytic activity of the associated CDK. Here we show that endogenous cyclin C protein is quite stable with a half-life of 4 h. In contrast, exogenously expressed cyclin C is very unstable (half-life 15 min) and degraded by the ubiquitin-proteasome pathway. Co-expression with its associated cdk, however, strongly stabilizes cyclin C and results in a protein half-life near that of endogenous cyclin C. In stark contrast to data reported for other members of the cyclin family, both catalytically active and inactive cdk8 induce cyclin C stabilization. Moreover, this stabilization is accompanied in both cases by phosphorylation of the cyclin, which is not detectable when unstable. Our results indicate that cyclin C has apparently diverged from other cyclins in the regulation of its stability by its CDK partner.

3T3 Cells↗
PubMed2000

Immunotherapy of a viral disease by in vivo production of therapeutic monoclonal antibodies.

Continuous and sustained in vivo production of monoclonal antibodies by engineered cells might render long-term antibody-based treatments cost-effective, avoid side effects associated with infusion of massive doses of antibody, and circumvent possible antiidiotypic responses against the therapeutic agent. The FrCasE retrovirus induces a lethal neurodegeneration on infection of newborn mice. We report here that implantation of cellulose sulfate capsules containing cells secreting an ectopic monoclonal antibody neutralizing FrCasE can prevent animals from developing the disease. All treated mice showed reduced or undetectable viremia in addition to a lack of the histopathological lesions characteristic of FrCasE infection. This work paves the way for a novel gene/cell antibody-based immunotherapy of a variety of severe viral and nonviral diseases.

Animals↗
PubMed2000

Antiviral activity of an intracellularly expressed single-chain antibody fragment directed against the murine leukemia virus capsid protein.

We have addressed the possibility that intracellularly expressed miniantibodies directed against the viral capsid protein can be used as antiretroviral agents in gene transfer experiments. R187 is a rat monoclonal antibody that has been reported to recognize the MuLV p30gag capsid polypeptide. We report here that it also binds to the Pr65gag precursor polyprotein. R187 has been cloned and expressed in the form of a single-chain variable fragment (scFv) that shows the same binding specificity as the parental antibody. When expressed intracellularly, the R187 scFv favors the production of viral particles showing reduced infectivity. It, however, exerts no detectable protective effect against infection. This was observed both when using replication-incompetent MuLV-derived vector and replication-competent wild-type MuLV. Although the intimate mechanism of the inhibition is not clear, this work raises the possibility that gene engineering of anti-capsid protein scFvs may offer an additional lead for gene therapy of severe retrovirus-linked diseases.

Amino Acid Sequence↗
PubMed2000

The sensitivity of c-Jun and c-Fos proteins to calpains depends on conformational determinants of the monomers and not on formation of dimers.

Milli- and micro-calpains are ubiquitous cytoplasmic cysteine proteases activated by calcium. They display a relatively strict specificity for their substrates which they usually cleave at only a limited number of sites. Motifs responsible for recognition by calpains have not been characterized yet, and recently a role for PEST motifs in this process has been ruled out. c-Fos and c-Jun transcription factors are highly sensitive to calpains in vitro. They thus provide favourable protein contexts for studying the structural requirements for recognition and degradation by these proteases. Using in vitro degradation assays and site-directed mutagenesis, we report here that susceptibility to calpains is primarily determined by conformational determinants of the monomers and not by the quaternary structure of c-Fos and c-Jun proteins. The multiple cleavage sites borne by both proteins can be divided into at least two classes of sensitivity, the most sensitive ones being easily visualized in the presence of rate-limiting amounts of calpains. One site located at position 90-91 in c-Fos protein is extremely sensitive. However, efficient proteolysis did not have any strict dependence on the nature of the amino acids on either side of the scissile bond in the region extending from P2 to P'2. The structural integrity of the monomers is not crucial for recognition by calpains. Rather, sensitive sites can be recognized independently and their recognition is dependent on the local conformation of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.

Amino Acid Sequence↗
PubMed2000

Molecular characterization of the thermosensitive E1 ubiquitin-activating enzyme cell mutant A31N-ts20. Requirements upon different levels of E1 for the ubiquitination/degradation of the various protein substrates in vivo.

According to our current knowledge, protein ubiquitination involves three steps: activation of ubiquitin through formation of an energy-rich bond with an E1 ubiquitin-activating enzyme; and transfer of activated ubiquitin onto E2 ubiquitin-conjugating enzymes, which, in turn, alone, or in combination with E3 ubiquitin-protein ligase enzymes, transfer ubiquitin onto target proteins. A31N-ts20 cells are mouse embryo fibroblasts, thermosensitive for E1. We show here that: (a) the enzymatic activity of the enzyme is heat-inactivatable in vitro; and (b) a major mechanism responsible for E1 inactivation in vivo consists of accelerated destruction. Surprisingly, a >90% reduction in E1 abundance little alters the formation of the bulk of protein-ubiquitin conjugates when A31N-ts20 cells are grown at the nonpermissive temperature, indicating that cautious interpretation of results is required when studying ubiquitination of specific substrates using this cell line. Surprisingly, our data also indicate that, in vivo, ubiquitination of the various protein substrates in A31N-ts20 cells requires different amounts of E1, indicating that this mutant cell line can be used for unveiling the existence of differences in the intimate mechanisms responsible for the ubiquitination of the various cell proteins in vivo, and for providing criteria of reliability when developing in vitro ubiquitination assays for specific proteins.

Animals↗
PubMed2000

Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts.

In vivo production and systemic delivery of therapeutic antibodies by engineered cells might advantageously replace injection of purified antibodies for treating a variety of life-threatening diseases, including cancer, acquired immunodeficiency syndrome, and autoimmune diseases. We report here that skin fibroblasts retrovirally transduced to express immunoglobulin genes can be used for sustained long-term systemic delivery of cloned antibodies in immunocompetent mice. Importantly, no anti- idiotypic response against the ectopically expressed model antibody used in this study was observed. This supports the notion that skin fibroblasts can potentially be used in antibody-based gene/cell therapy protocols without inducing any adverse immune response in treated individuals.

Animals↗
PubMed2000

Efficient cell infection by Moloney murine leukemia virus-derived particles requires minimal amounts of envelope glycoprotein.

Retrovirus entry into cells is mediated by specific interactions between the retrovirally encoded Env envelope glycoprotein and a host cell surface receptor. Though a number of peptide motifs responsible for the structure as well as for the binding and fusion activities of Env have been identified, only a few quantitative data concerning the infection process are available. Using an inducible expression system, we have expressed various amounts of ecotropic and amphotropic Env at the surfaces of Moloney murine leukemia virus-derived vectors and assayed for the infectivity of viral particles. Contrary to the current view that numerous noncooperative Env-viral receptor interactions are required for cell infection, we report here that very small amounts of Env are sufficient for optimal infection. However, increasing Env density clearly accelerates the rate at which infectious attachment to cells occurs. Moreover, our data also show that a surprisingly small number of Env molecules are sufficient to drive infection, albeit at a reduced efficiency, and that, under conditions of low expression, Env molecules act cooperatively. These observations have important consequences for our understanding of natural retroviral infection as well as for the design of cell-targeted infection techniques involving retroviral vectors.

3T3 Cells↗
PubMed1999

Development of cellulose sulfate-based polyelectrolyte complex microcapsules for medical applications.

Microencapsulation, as a tool for immunoisolation for allogenic or xenogenic implants, is a rapidly growing field. However most of the approaches are based on alginate/polylysine capsules, despite this system's obvious disadvantages such as its pyrogenicity. Here we report a different encapsulation system based on sodium cellulose sulfate and polydiallyldimethyl ammonium chloride for the encapsulation of mammalian cells. We have characterized this system regarding capsule formation, strength and size of the capsules as well as viability of the cells after encapsulation. In addition, we demonstrate the efficacy of these capsules as a "microfactory" in vitro and in vivo. Using encapsulated hybridoma cells we were able to demonstrate long-term release of antibodies up to four months in vivo. In another application we could show the therapeutic relevance of encapsulated genetically modified cells as an in vivo activation center for cytostatic drugs during tumor therapy.

Alginates↗
PubMed1999

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.

Animals↗
PubMed1999

Are there multiple proteolytic pathways contributing to c-Fos, c-Jun and p53 protein degradation in vivo?

The c-Fos and c-Jun oncoproteins and the p53 tumor suppressor protein are short-lived transcription factors. Several catabolic pathways contribute to their degradation in vivo. c-Fos and c-Jun are thus mostly degraded by the proteasome, but there is indirect evidence that, under certain experimental/physiological conditions, calpains participate in their destruction, at least to a limited extent. Lysosomes have also been reported to participate in the destruction of c-Fos. Along the same lines, p53 is mostly degraded following the ubiquitin/proteasome pathway and calpains also seem to participate in its degradation. Moreover, c-Fos, c-Jun and p53 turnovers are regulated upon activation of intracellular signalling cascades. All taken together, these observations underline the complexity of the mechanisms responsible for the selective destruction of proteins within cells.

Animals↗
PubMed1998

Genetically engineered antibodies in gene transfer and gene therapy.

Our ability to produce and engineer human monoclonal antibodies provides a basis for the development of novel therapeutical strategies against a variety of diseases. These strategies not only include improved passive immunotherapy but also more sophisticated antibody-based gene therapies involving gene transfer approaches. Four of the major applications of antibody gene engineering in the field of gene therapy are reviewed here. These are (1) the redefinition of viral vector tropism of infection for better transduction of cells of therapeutical interest, (2) the grafting of new cell recognition activities to effector cells of the immune system to kill cancer and pathogen-infected cells, (3) the inhibition of cellular and viral functions through intracellular expression of antibody-derived molecules, and (4) the systemic delivery of therapeutic monoclonal antibodies by non-B cells in living organisms.

Antibodies, Monoclonal↗
PubMed1998

Differential directing of c-Fos and c-Jun proteins to the proteasome in serum-stimulated mouse embryo fibroblasts.

c-Fos and c-Jun proteins are highly unstable transcription factors that heterodimerize within the AP-1 transcription complex. Their accumulation is transiently induced at the beginning of the G0-to-S phase transition in quiescent cells stimulated for growth. To address the mechanisms responsible for rapid clearance of c-Fos and c-Jun proteins under these experimental conditions, we have used the ts20 mouse embryo fibroblasts which express a thermosensitive mutant of the E1 enzyme of the ubiquitin pathway. The use of cell-permeant protease inhibitors indicates that both proteins are degraded by the proteasome and excludes any major contribution for calpains and lysosomes during the G0-to-S phase transition. Synchronisation of ts20 cells at the non permissive temperature blocks the degradation of c-Jun, indicating that this process is E1-dependent. In contrast, c-Fos is broken down according to an apparently E1-independent pathway in ts20 cells, although a role for ubiquitinylation in this process cannot be formally ruled out. Interestingly, c-Jun is highly unstable in c-Fos-null mouse embryo fibroblasts stimulated for growth. Taken together, these observations show that in vivo during a G0-to-S phase transition (i) the precise mechanisms triggering c-Fos and c-Jun directing to the proteasome are not identical, (ii) the presence of c-Fos is not an absolute prerequisite for the degradation of c-Jun and (iii) the degradation of c-Jun is not required for that of c-Fos.

3T3 Cells↗
PubMed1998

Affinity of recombinant antibody and antibody fragment binding to human thyroglobulin: potential applications in gene therapy.

Recombinant antibodies and antibody fragments are currently being produced. They can be used in vitro for the structural study of antigen-antibody interactions for instance, but their in vivo production may have applications for gene therapy of certain cancers and severe viral diseases and in developing new animal models of autoimmune disease. We report here these two types of applications using a recombinant antihuman thyroglobulin (hTg) antibody.

Animals↗
PubMed1998

Cell targeting by murine retroviral vectors.

Gene therapy involves the transfer of new genetic material to cells of individuals with the aim of conferring a therapeutical benefit. Theoretically, gene therapy can be used to treat a variety of life-threatening disorders, such as inherited genetic diseases, cancer, chronic viral infections as well as a number of other severe diseases which are not treatable at present. To this aim, both efficient gene delivery techniques and controlled gene expression systems are required. Engineered murine retroviruses are the most widely used vectors for stable clinical gene transfer because of their ability to integrate--along with the transgenes they are engineered to carry--into the genome of infected cells. However, this technology still suffers from a number of drawbacks and limitations. Particularly, the ability to target cells of therapeutic interest together with the controlled expression of transferred genes would improve both the efficiency and the safety of retroviral vectors. Such improvements would additionally allow the development of new animal models of human diseases as well as enable more fundamental investigations in the fields of oncogenesis and developmental biology.

Animals↗
PubMed1998

Systemic long-term delivery of antibodies in immunocompetent animals using cellulose sulphate capsules containing antibody-producing cells.

Implantation of capsules containing antibody-producing cells into patients would potentially permit systemic long-term delivery of antibodies and might, thus, be useful in the development of surveillance treatments for cancers and severe viral diseases. We show that cellulose sulphate (CS) capsules containing hybridoma cells, when implanted subcutaneously or in the intraperitoneal cavity, can be used for delivering monoclonal antibodies into the blood-stream of immunocompetent mice for at least several months. In contrast to capsules implanted into the intraperitoneal cavity, which remain mobile and nonvascularized, capsules implanted under the skin form neo-organs which become vascularized within days. This may explain the higher blood concentration of the antibody we have observed in the latter case. Importantly, neither an isolating fibrosis nor an obvious inflammatory response was detected at the capsule implantation sites during observation periods as long as 10 months. Finally, no anti-idiotypic immune response against the ectopically delivered antibody was shown to occur. This rules out any potent adjuvant effect of the cellulose sulphate matrix that might have stimulated a neutralizing humoral response. Taken together, our data indicate that encapsulation of antibody-producing cells into CS might be used in antibody-based gene/cell therapy approaches.

Animals↗