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Tag: Pellegrini

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PLoS One. 2015 Nov 3;10(11):e0141692. doi: 10.1371/journal.pone.0141692. eCollection 2015.

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin.

Gaidos G1, Panaitiu AE1, Guo B1, Pellegrini M1, Mierke DF1.
Fig 6. Model of R4 peptide/calmodulin complex.

Abstract

Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.

http://www.ncbi.nlm.nih.gov/pubmed/26529318

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Scientific Reports 5, Article number: 9893 doi:10.1038/srep09893

A novel caspase 8 selective small molecule potentiates TRAIL-induced cell death

Octavian Bucur, Gabriel Gaidos, Achani Yatawara, Bodvael Pennarun, Chamila Rupasinghe, Jérémie Roux, Stefan Andrei, Bingqian Guo, Alexandra Panaitiu, Maria Pellegrini, Dale F. Mierke & Roya Khosravi-Far

CasPro

Recombinant soluble TRAIL and agonistic antibodies against TRAIL receptors (DR4 and DR5) are currently being created for clinical cancer therapy, due to their selective killing of cancer cells and high safety characteristics. However, resistance to TRAIL and other targeted therapies is an important issue facing current cancer research field. An attractive strategy to sensitize resistant malignancies to TRAIL-induced cell death is the design of small molecules that target and promote caspase 8 activation. For the first time, we describe the discovery and characterization of a small molecule that directly binds caspase 8 and enhances its activation when combined with TRAIL, but not alone. The molecule was identified through an in silico chemical screen for compounds with affinity for the caspase 8 homodimer’s interface. The compound was experimentally validated to directly bind caspase 8, and to promote caspase 8 activation and cell death in single living cells or population of cells, upon TRAIL stimulation. Our approach is a proof-of-concept strategy leading to the discovery of a novel small molecule that not only stimulates TRAIL-induced apoptosis in cancer cells, but may also provide insights into the structure-function relationship of caspase 8 homodimers as putative targets in cancer.

http://www.nature.com/srep/2015/150511/srep09893/full/srep09893.html

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J Biol Chem. 2015 290(5):2879-87.  doi: 10.1074/jbc.M114.609768. PMID: 25492869

"Phosphorylation of Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) by Akt Promotes Stability and Mitogenic Function of S-phase Kinase-associated Protein-2 (Skp2)."

Song GJ, Leslie KL, Barrick S, Mamonova T, Fitzpatrick JM, Drombosky KW, Peyser N, Wang B, Pellegrini M, Bauer PM, Friedman PA, Mierke DF, Bisello A.  

Abstract

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.

 

 

Published on
J Pept Sci. 2014 Dec 19. doi: 10.1002/psc.2731. [Epub ahead of print]

Small-molecule inhibitors of JC polyomavirus infection.

Yatawara A1, Gaidos G, Rupasinghe CN, O'Hara BA, Pellegrini M, Atwood WJ, Mierke DF.

Abstract

The JC polyomavirus (JCPyV) infects approximately 50% of the human population. In healthy individuals, the infection remains dormant and asymptomatic, but in immuno-suppressed patients, it can cause progressive multifocal leukoencephalopathy (PML), a potentially fatal demyelinating disease. Currently, there are no drugs against JCPyV infection nor for the treatment of PML. Here, we report the development of small-molecule inhibitors of JCPyV that target the initial interaction between the virus and host cell and thereby block viral entry. Utilizing a combination of computational and NMR-based screening techniques, we target the LSTc tetrasaccharide binding site within the VP1 pentameric coat protein of JCPyV. Four of the compounds from the screen effectively block viral infection in our in vitro assays using SVG-A cells. For the most potent compound, we used saturation transfer difference NMR to determine the mode of binding to purified pentamers of JCPyV VP1. Collectively, these results demonstrate the viability of this class of compounds for eventual development of JCPyV-antiviral therapeutics.

http://www.ncbi.nlm.nih.gov/pubmed/25522925

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Biochemistry. 2014  Nov 4;53(43):6776-85. doi: 10.1021/bi500861x. Epub 2014 Oct 23

Protein Engineering of the N-terminus of NEMO: structure stabilization and rescue of IKKβ binding.

Abstract

NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKβ, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-ĸB pathway. Rational drug design targeting the IKK binding site on NEMO would benefit from structural insight, but to date the structure determination of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK binding domain NEMO(44-111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled-coil at the N-terminus, C-terminus or both ends of NEMO(44-111) present high thermal stability and cooperative melting, and most importantly restore IKKß binding affinity. We examined the consequences on structural content and stability by circular dichoism and nuclear magnetic resonance and measured binding affinity of each construct for IKKβ(701-745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method to engineer short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile the rescuing of the binding affinity implies that a pre-ordered IKK-binding region of NEMO is compatible with IKK binding and the conformational heterogeneity observed in NEMO(44-111) may be an artifact of the truncation.

http://www.ncbi.nlm.nih.gov/pubmed/25286246

C-terminal coiled-coil adaptor fused to NEMO
C-terminal coiled-coil adaptor fused to NEMO

 

Published on
Biochemistry. 2014 Sep 23;53(37):5916-22. doi: 10.1021/bi500368k. Epub 2014 Sep 15.

Small Molecule Inhibition of the Na+/H+ Exchange Regulatory Factor 1 and Parathyroid Hormone 1 Receptor Interaction.

Abstract

We have identified a series of small molecules that bind to the canonical peptide binding groove of the PDZ1 domain of NHERF1 and effectively compete with the association of the C-terminus of the parathyroid hormone 1 receptor (PTH1R). Employing nuclear magnetic resonance and molecular modeling, we characterize the mode of binding that involves the GYGF loop important for the association of the C-terminus of PTH1R. We demonstrate that the common core of the small molecules binds to the PDZ1 domain of NHERF1 and displaces a 15N-labeled peptide corresponding to the C-terminus of PTH1R. The small size (molecular weight of 192) of this core scaffold makes it an excellent candidate for further elaboration in the development of an inhibitor for this important protein-protein interaction.

http://www.ncbi.nlm.nih.gov/pubmed/25171053

Small molecule docked to NHERF PDZ
Small molecule docked to NHERF PDZ

Published on
ATP_paper_artChem Commun (Camb). 2014 Sep 16;50(81):12037-9. doi: 10.1039/c4cc04399e.

Monitoring ATP hydrolysis and ATPase inhibitor screening using (1)H NMR.

Abstract

We present a versatile method to characterize ATPase and kinase activities and discover new inhibitors of these proteins. The proton NMR-based assay directly monitors ATP turnover and is easy to implement, requires no additional reagents and can potentially be applied to GTP. We validated the method's accuracy, applied it to the monitoring of ATP turnover by actin and to the screening of ATPase inhibitors, and showed that it is also applicable for the monitoring of GTP hydrolysis.

http://www.ncbi.nlm.nih.gov/pubmed/25170530