Biology subjects

Zenkeviciute, G.

Publications and source records attributed to Zenkeviciute, G..

3 recordsLinked to original sources

bioRxiv2023

Development of Constrained Peptide Inhibitors Targeting an Oncogenic E3 Ubiquitin Ligase

SCFSkp2/Cks1 is an E3 ubiquitin ligase, whose substrate specificity is determined by the oncogenic F-box protein Skp2 and the adaptor protein Cks1. A principal target of SCFSkp2/Cks1 is the cyclin-dependent kinase inhibitor p27. Elevated levels of Skp2 and reduced levels of p27 are common in a variety of cancers, and there is consequently a need to develop effective inhibitors of the Skp2-p27 interaction. However, conventional small-molecule approaches are challenging due to the extended bi-molecular interface that spans both Skp2 and Cks1, the lack of suitable binding pockets on this surface, and the intrinsically disordered nature of p27. Here, we develop macrocyclic peptides capable of binding to SCFSkp2/Cks1 with nanomolar affinities, an enhancement of almost two orders of magnitude over the natural p27 peptide. We show that these macrocyclic peptides inhibit p27 ubiquitination in vitro, restore p27 levels in a breast cancer cell line, and reduce cell proliferation.

cancer biology↗

bioRxiv2021

Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a

We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 plays a critical role in the cell cycle by inhibiting cyclin-dependent kinases CDK4 and CDK6, and it is inactivated in sporadic and familial cancers. The majority of the p16 missense mutations cause loss of function by destabilizing the proteins structure. We show that the nanobodies bind p16 with nanomolar affinities and restore the stability of a range of different cancer-associated p16 mutations located at sites throughout the protein. The nanobodies also bind and stabilize p16 in a cellular setting. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. These findings indicate that nanobodies could be used as pharmacological chaperones to determine the consequences of restoring the function of p16 in the cell. HighlightsO_LIDescribes a nanobody (single-domain antibody) capable of binding tumor suppressor protein p16 C_LIO_LINanobody binding can stabilise wild-type and cancer-associated p16 mutants C_LIO_LINanobody-p16 crystal structure reveals why nanobody binding is compatible with p16-CDK6 interaction C_LI

biochemistry↗

bioRxiv2018

Structural variability of EspG chaperones from mycobacterial ESX-1, ESX-3 and ESX-5 type VII secretion systems

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central {beta}-sheet and two -helical bundles. Additionally, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3 and EspG5 chaperones using small-angle X-ray scattering (SAXS) reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the SAXS analysis.\n\nHighlightsO_LIThe crystal structure of EspG1 reveals the common architecture of the type VII secretion system chaperones\nC_LIO_LIStructures of EspG3 chaperones display a number of conformations that could reflect alternative substrate binding modes\nC_LIO_LIEspG3 chaperones dimerize in solution\nC_LIO_LIA model of EspG3 in complex with its substrate PE-PPE dimer is proposed based on SAXS data\nC_LI

biochemistry↗