Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops

Large scale expression and purification of full-length huntingtin Q23 with HAP40 from baculoviral expression system production in sf9 insect cells – 2018/05/28

Project - Huntingtin structure-function open lab notebook. Experiment and rationale - Purified huntingtin samples with stabilising binding partners (e.g. HAP40) are required for use in structural and functional studies, in particular SAXS experiments.Dr. Harding is the recipient of the Huntington's Disease Society of America Berman Topper Career Development Fellowship which funds and supports this research. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome

Purification of full-length huntingtin Q23 with C-terminal FLAG tag (2017/07/19)

Huntingtin structure-function open lab notebook projec

Full-length huntingtin Q23 expression and purification – 19th November 2017

Huntingtin structure-function open lab noteboo

Expression and purification of huntingtin domain constructs spanning aa. P80-G428 – 2017/08/09

Huntingtin structure-function open lab notebook projec

Full-length HTT purification from HEK293T suspension culture using baculovirus transduction protocol for over-expression 2018/04/02

<p>Structure-function open lab notebook project. Full-length HTT purification from HEK293T suspension culture using baculovirus transduction protocol for over-expression 2018/04/02.</p

Large-scale full-length HTT Q19 and Q42 purification from HEK293T – 2018/04/16

<p>Huntingtin structure-function open lab notebook project. Large-scale full-length HTT Q19 and Q42 purification from HEK293T – 2018/04/16.</p

Cloning, eukaryotic expression and purification of full-length huntingtin Q23 (2017/06/01)

Huntingtin structure function open lab notebook projec

Small scale and large scale purification of full-length huntingtin Q17 and Q46 from mammalian expression system (2016/11/29)

Huntingtin structure function open lab notebook projec