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Biomedical subjects

May C Morris

Publications and source records attributed to May C Morris.

13 recordsLinked to original sources
PubMed2007

The alpha helix of ubiquitin interacts with yeast cyclin-dependent kinase subunit CKS1.

Ubiquitin serves as a molecular zipcode to direct and sort ubiquitinylated proteins into distinct biological pathways. Although novel modes of ubiquitin interaction have recently been characterized, conventional ubiquitin-binding domains (UBDs) recognize ubiquitin through a hydrophobic pocket centered around isoleucine 44 and lined by residues in beta sheets 3 and 4. In this study, we report a novel mode of interaction between ubiquitin and the cyclin-dependent kinase subunit of Saccharomyces cerevisiae, Cks1p, an adaptor protein involved in transcriptional regulation through recruitment of proteasomal subunits to gene promoters. Cks1p interacts specifically with monoubiquitin and tetraubiquitin with an affinity several orders of magnitude greater than that of other ubiquitin-binding domains and in an unconventional fashion, which differs from interactions documented so far between ubiquitin and conventional UBDs. The loop between helices alpha 1 and alpha 2, and to a minor extent the N-terminal alpha-helix of Cks1p, are involved in the interaction with the alpha-helix of ubiquitin, instead of its I44-centered hydrophobic pocket. Not only is this the first time the alpha-helix of ubiquitin is implicated in a protein/protein interaction, thereby shedding new light on the mechanisms of ubiquitin recognition, but also the first report of a direct physical interaction between ubiquitin and Cks1p, inferring a role for ubiquitin binding in the transcriptional function of Cks1p.

Adaptor Proteins, Signal Transducing↗
PubMed2006

A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction.

The development of therapeutic peptides and proteins is limited by the poor permeability and the selectivity of the cell membrane. The discovery of protein transduction domains has given a new hope for administration of large proteins and peptides in vivo. We have developed a non-covalent strategy for protein transduction based on an amphipathic peptide, Pep-1, that consists of a hydrophobic domain and a hydrophilic lysine-rich domain. Pep-1 efficiently delivers a variety of fully biologically active peptides and proteins into cells, without the need for prior chemical cross-linking or chemical modifications. The mechanism through which Pep-1 delivers active macromolecules does not involve the endosomal pathway and the dissociation of the Pep-1/macromolecule particle occurs immediately after it crosses the cell membrane. Pep-1 has been successfully applied to the screening of therapeutic peptides in vivo and presents several advantages: stability in physiological buffer, lack of toxicity and of sensitivity to serum. In conclusion, Pep-1 technology could contribute significantly to the development of fundamental and therapeutic applications and be an alternative to covalent protein transduction domain-based technologies.

Animals↗
PubMed2006

Interactions of amphipathic carrier peptides with membrane components in relation with their ability to deliver therapeutics.

To identify rules for the design of efficient CPPs that can deliver therapeutic agents such as nucleic acids (DNAs, siRNAs) or proteins and PNAs into subcellular compartments, we compared the properties of several primary and secondary amphipathic CPPs. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interactions govern the conformational state(s) of the peptides. On the basis of the ability of both peptides to promote ion permeation through both natural and artificial membranes, models illustrating the translocation processes have been proposed. One is based on the formation of a beta-barrel pore-like structure while another is based on the association of helices.

Amino Acid Sequence↗
PubMed2005

Interactions of amphipathic CPPs with model membranes.

We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a beta-barrel pore-like structure, while for Pep-1, it is based on association of helices.

Amino Acid Sequence↗
PubMed2005

Design of a novel class of peptide inhibitors of cyclin-dependent kinase/cyclin activation.

Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.

Amino Acid Sequence↗
PubMed2005

Interactions of primary amphipathic cell penetrating peptides with model membranes: consequences on the mechanisms of intracellular delivery of therapeutics.

This review focuses on two peptides, MPG and Pep-1, which facilitate the transfer of nucleic acids and proteins, respectively, into subcellular compartments. We have investigated the interactions between these two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG it is based on the formation of a beta-barrel pore-like structure while for Pep-1 it is based on association of helices.

Amino Acid Sequence↗
PubMed2004

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids.

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a beta-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.

Amino Acid Sequence↗
PubMed2004

Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis.

Recently, we described a new strategy for the delivery of proteins and peptides into mammalian cells, based on an amphipathic peptide of 21 residues, Pep-1, which was designed on the basis of a protein-interacting domain associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of active proteins or peptides both in cultured cells and in vivo, without requiring any covalent coupling. We have examined the conformational states of Pep-1 in its free form and complexed with a cargo peptide and have investigated their ability to interact with phospholipids and the structural consequences of these interactions. From the conformational point of view, Pep-1 behaves significantly differently from other similarly designed cell-penetrating peptides. CD analysis revealed a transition from a nonstructured to a helical conformation upon increase of the concentration. Determination of the structure by NMR showed that in water, its alpha-helical domain extends from residues 4-13. CD and FTIR indicate that Pep-1 adopts a helical conformation in the presence of phospholipids. Adsorption measurements performed at the air-water interface are consistent with the helical form. Pep-1 does not undergo conformational changes upon formation of a particle with a cargo peptide. In contrast, we observe a partial conformational transition when the complex encounters phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Conformational change of Pep-1 is not associated with complexation with its cargo but is induced upon association with the cell membrane.

Air↗
PubMed2003

Cks1-dependent proteasome recruitment and activation of CDC20 transcription in budding yeast.

Cks proteins are small evolutionarily conserved proteins that interact genetically and physically with cyclin-dependent kinases. However, in spite of a large body of genetic, biochemical and structural research, no compelling unifying model of their functions has emerged. Here we show, by investigating the essential role of Cks1 in Saccharomyces cerevisiae, that the protein is primarily involved in promoting mitosis by modulating the transcriptional activation of the APC/C protein-ubiquitin ligase activator Cdc20. Cks1 is required for both the periodic dissociation of Cdc28 kinase from the CDC20 promoter and the periodic association of the proteasome with the promoter. We propose that the essential role of Cks1 is to recruit the proteasome to, and/or dissociate the Cdc28 kinase from, the CDC20 promoter, thus facilitating transcription by remodelling transcriptional complexes or chromatin associated with the CDC20 gene.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells.

The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.

Active Transport, Cell Nucleus↗
PubMed2003

Functional cdc25C dual-specificity phosphatase is required for S-phase entry in human cells.

In view of the common regulatory mechanism that induces transcription of the mitotic phosphatase cdc25C and cyclin A at the beginning of S-phase, we investigated whether cdc25C was required for S-phase transit. Here, we show that in both nontransformed human fibroblasts and HeLa cells, cdc25C protein levels significantly increased concomitant with S-phase onset and cyclin A synthesis. Activity measurements on immunoprecipitates from synchronized HeLa cells revealed a sharp rise in cdc25C-associated phosphatase activity that coincided with S-phase. Microinjection of various antisense-cdc25C molecules led to inhibition of DNA synthesis in both HeLa cells and human fibroblasts. Furthermore, transfection of small interfering RNA directed against cdc25C specifically depleted cdc25C in HeLa cells without affecting cdc25A or cdc25B levels. Cdc25C RNA interference was also accompanied by S-phase inhibition. In cells depleted of cdc25C by antisense or siRNA, normal cell cycle progression could be re-established through microinjection of wild-type cdc25C protein but not inactive C377S mutant protein. Taken together, these results show that cdc25C not only plays a role at the G2/M transition but also in the modulation of DNA replication where its function is distinct from that of cdc25A.

Cell Cycle↗
PubMed2002

Kinetic mechanism of activation of the Cdk2/cyclin A complex. Key role of the C-lobe of the Cdk.

Eukaryotic cell cycle progression is controlled by the ordered action of cyclin-dependent kinases, activation of which occurs through the binding of the cyclin to the Cdk followed by phosphorylation of a conserved threonine in the T-loop of the Cdk by Cdk-activating kinase (CAK). Despite our understanding of the structural changes, which occur upon Cdk/cyclin formation and activation, little is known about the dynamics of the molecular events involved. We have characterized the mechanism of Cdk2/cyclin A complex formation and activation at the molecular and dynamic level by rapid kinetics and demonstrate here that it is a two-step process. The first step involves the rapid association between the PSTAIRE helix of Cdk2 and helices 3 and 5 of the cyclin to yield an intermediate complex in which the threonine in the T-loop is not accessible for phosphorylation. Additional contacts between the C-lobe of the Cdk and the N-terminal helix of the cyclin then induce the isomerization of the Cdk into a fully mature form by promoting the exposure of the T-loop for phosphorylation by CAK and the formation of the substrate binding site. This conformational change is selective for the cyclin partner.

Amino Acid Motifs↗