Biology subjects

Pantelejevs, T.

Publications and source records attributed to Pantelejevs, T..

6 recordsLinked to original sources

bioRxiv2023

Structural basis of epitope recognition by anti-alpha synuclein antibodies MJFR14-6-4-2

Intraneuronal -synuclein inclusions in the brain are hallmarks of so-called Lewy body diseases - Parkinsons disease and Dementia with Lewy bodies. Lewy bodies are cytoplasmic inclusions, containing mainly aggregated -synuclein together with some other proteins including ubiquitin, neurofilament protein, and alpha B crystallin. In its monomeric form, -synuclein is predominantly localized in nerve terminals, regulating neuronal transmission and synaptic vesicle trafficking. Monomeric -synuclein lacks a well-defined three-dimensional structure and is considered an intrinsically disordered protein. However, in diseased cells -synuclein aggregates into oligomeric and fibrillar amyloid species, which can be detected using aggregate-specific antibodies. Here we investigate the aggregate specificity of rabbit monoclonal MJFR14-6-4-2 antibodies, preferentially recognizing aggregated -synuclein species. We conclude that partial masking of epitope in unstructured monomer in combination with a high local concentration of epitopes instead of distinct epitope conformation is the main reason for apparent selectivity towards various aggregates, including oligomers, fibrils, and artificial virus-like particle constructs bearing multiple copies of the MJFR14-6-4-2 epitope. Based on the structural insight, we were able to express mutant -synuclein that when fibrillated are unable to bind MJFR14-6-4-2. Using these "stealth" fibrils as a tool for seeding cellular -synuclein aggregation, provides superior signal/noise ratio for detection of cellular -synuclein aggregates by MJFR14-6-4-2 immunocytochemistry. Our data provide a molecular level understanding of specific recognition of toxic amyloid oligomers, which is critical for the development of inhibitors against synucleinopathies.

neuroscience↗

bioRxiv2023

A Recombinant Approach For Stapled Peptide Discovery YieldsInhibitors of the RAD51 Recombinase

Stapling is a macrocyclisation method that connects amino acid side chains of a peptide to improve its pharmacological properties. We describe an approach for stapled peptide preparation and biochemical evaluation that combines recombinant expression of fusion constructs of target peptides and cysteine-reactive divinyl-heteroaryl chemistry, as an alternative to solid-phase synthesis. We then employ this workflow to prepare and evaluate BRC-repeat-derived inhibitors of the RAD51 recombinase, showing that a diverse range of secondary structure elements in the BRC repeat can be stapled without compromising binding and function. Using X-ray crystallography, we elucidate the atomic-level features of the staple moieties. We then demonstrate that BRC-repeat-derived stapled peptides can disrupt RAD51 function in cells following ionising radiation treatment.

biochemistry↗

bioRxiv2022

Targeting triple-negative breast cancer with β1-integrin binding aptamer

Targeted therapies have increased the treatment options for triple-negative breast cancer patients. However, the paucity of targetable biomarkers and tumour heterogeneity have limited the ability of precision-guided interventions to live up to their full potential. As affinity targeting ligands, aptamers show high selectivity towards target molecules. Compared to antibodies, aptamers have lower molecular weight, increased stability during transportation, reduced immunogenicity, and increased tissue uptake. Recently, we reported the discovery of GreenB1 aptamer that is internalized in cultured triple-negative MDA-MB-231 human breast cancer cells. We show that the GreenB1 aptamer specifically targets {beta}1-integrin, a protein previously linked to breast cancer cell invasiveness and migration. Aptamer binds to {beta}1-integrin with low nanomolar affinity. GreenB1 homes in the orthotopic 4T1 triple-negative breast cancer lesions modelled in mice. Our findings suggest potential applications for the GreenB1-guided precision agents for the diagnosis and therapy of triple-negative breast cancer.

molecular biology↗

bioRxiv2022

Divergent binding mode for a protozoan BRC repeat to RAD51

Interaction of BRCA2 through ca. 30 amino acid residue motifs, BRC repeats, with RAD51 is a conserved feature of the double-strand DNA break repair process in eukaryotes. In humans the binding of the eight BRC repeats is relatively well understood, with structure of BRC4 repeat bound to human RAD51 showing how two sequence motifs, FxxA and LFDE, in the BRC repeat interact with distinct sites on RAD51. Little is known however of the interaction of BRC repeats in other species, especially in protozoans where variable number of BRC repeats are found in BRCA2 proteins. Here we have studied in detail the interactions of the two BRC repeats in Leishmania infantum BRCA2 with RAD51. We show that the LiBRC1 is a high affinity repeat with a KD of 0.29 M while LiBRC2 binds to RAD51 with a KD of 13.5 M. A crystal structure of LiBRC1 complexed with LiRAD51 revels an extended {beta}-hairpin compared to human BRC4 and shows that the equivalent of human LFDE motif is not interacting with LiRAD51. A truncation analysis of LiBRC1 confirms that a shorter repeat is sufficient for high affinity interaction and this minimal repeat is functional in inhibiting the formation of LiRAD51-ssDNA nucleofilament.

biochemistry↗

bioRxiv2021

A strategy to suppress STAT1 signalling conserved in pathogenic poxviruses and paramyxoviruses

The induction of interferon-stimulated genes by signal transducer and activator of transcription (STAT) proteins, is a critical host defence to fight virus infections. Here, a highly expressed poxvirus protein 018 is shown to inhibit IFN-induced signalling by binding the SH2 domain of STAT1 to prevent STAT1 association with an activated IFN receptor. Despite the presence of additional inhibitors of IFN-induced signalling, a poxvirus lacking 018 was attenuated in mice. The 2.0 [A] crystal structure of the 018:STAT1 complex reveals a mechanism for a high-affinity, pTyr-independent mode of binding to an SH2 domain. Furthermore, the STAT1 binding motif of 018 shows sequence similarity to the STAT1-binding proteins from Nipah virus, which like 018, block the association of STAT1 with an IFN receptor. Taken together, these results provide detailed mechanistic insight into a potent mode of STAT1 antagonism, found to exist in genetically diverse virus families.

microbiology↗

bioRxiv2020

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled repeats were stronger than any of the natural repeats, suggesting balancing of relative properties in BRC repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.44 kCal/mol compared to the strongest natural repeat, BRC4. Crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended {beta}-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear foci after ionizing radiation.

biochemistry↗