Exploiting the MDM2-CK1 alpha Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α) forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2) oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i) ELISA with recombinant MDM2; (ii) cell lysate pull-down towards endogenous MDM2; (iii) MDM2-CK1α complex-based competition ELISA; and (iv) MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i) function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii) be used as a tool to study NEDDylation of CK1α, and (iii) reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross-talk between NEDDylation, protein kinase signalling, and cell survival

p21(Cip1) plays a critical role in the physiological adaptation to fasting through activation of PPARα.

Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the role of stress-responsive tumor suppressors in fasting. Here, we have examined the expression of several tumor suppressors upon fasting in mice. Interestingly, p21 mRNA is uniquely induced in all the tissues tested, particularly in liver and muscle (>10 fold), and this upregulation is independent of p53. Remarkably, in contrast to wild-type mice, p21-null mice become severely morbid after prolonged fasting. The defective adaptation to fasting of p21-null mice is associated to elevated energy expenditure, accelerated depletion of fat stores, and premature activation of protein catabolism in the muscle. Analysis of the liver transcriptome and cell-based assays revealed that the absence of p21 partially impairs the transcriptional program of PPARα, a key regulator of fasting metabolism. Finally, treatment of p21-null mice with a PPARα agonist substantially protects them from their accelerated loss of fat upon fasting. We conclude that p21 plays a relevant role in fasting adaptation through the positive regulation of PPARα

Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity

Diphenyl Urea Derivatives as Inhibitors of Transketolase: A Structure-Based Virtual Screening

Transketolase is an enzyme involved in a critical step of the non-oxidative branch of the pentose phosphate pathway whose inhibition could lead to new anticancer drugs. Here, we report new human transketolase inhibitors, based on the phenyl urea scaffold, found by applying structure-based virtual screening. These inhibitors are designed to cover a hot spot in the dimerization interface of the homodimer of the enzyme, providing for the first time compounds with a suggested novel binding mode not based on mimicking the thiamine pyrophosphate cofactor

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.This work was financed by grants BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness and 612146/FP7-ICT- 2013 and 282004/FP7-HEALTH.2011.2.3.1-2 from the European Union Seventh Framework Programme to FC and grant BFU2014-55534-C2-2-P from the Spanish Ministry of Economy and Competitiveness to GM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Assigning Backbone NMR Resonances for Full Length Tau Isoforms: Efficient Compromise between Manual Assignments and Reduced Dimensionality

Tau protein is the longest disordered protein for which nearly complete backbone NMR resonance assignments have been reported. Full-length tau protein was initially assigned using a laborious combination of bootstrapping assignments from shorter tau fragments and conventional triple resonance NMR experiments. Subsequently it was reported that assignments of comparable quality could be obtained in a fully automated fashion from data obtained using reduced dimensionality NMR (RDNMR) experiments employing a large number of indirect dimensions. Although the latter strategy offers many advantages, it presents some difficulties if manual intervention, confirmation, or correction of the assignments is desirable, as may often be the case for long disordered and degenerate polypeptide sequences. Here we demonstrate that nearly complete backbone resonance assignments for full-length tau isoforms can be obtained without resorting either to bootstrapping from smaller fragments or to very high dimensionality experiments and automation. Instead, a set of RDNMR triple resonance experiments of modest dimensionality lend themselves readily to efficient and unambiguous manual assignments. An analysis of the backbone chemical shifts obtained in this fashion indicates several regions in full length tau with a notable propensity for helical or strand-like structure that are in good agreement with previous observations

The p12 Domain Is Unstructured in a Murine Leukemia Virus p12-CAN Gag Construct

The Gag polyproteins of gammaretroviruses contain a conserved p12 domain between MA and CA that plays critical roles in virus assembly, reverse transcription and nuclear integration. Here we show using nuclear magnetic resonance, that p12 is unstructured in a Moloney murine leukemia virus (MMLV) Gag fragment that includes the N-terminal domain of CA (p12-CAN). Furthermore, no long range interactions were observed between the domains, as has been previously predicted. Flexibility appears to be a common feature of Gag “late” domains required for virus release during budding. Residues near the N-terminus of CAN that form a β-hairpin in the mature CA protein are unfolded in p12-CAN, consistent with proposals that hairpin formation helps trigger capsid assembly

Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that interact and dissociate the tubulin dimer. Here we show how TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO– present in TBCB, which is similar to the EEY/F-COO– element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE–TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated to microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation

Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

<p>Abstract</p> <p>Background</p> <p>In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses) via horizontal gene transfer (HGT). It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes.</p> <p>Results</p> <p>Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA) viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep)-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses.</p> <p>Conclusions</p> <p>Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.</p