Ingrid Dreveny’s research while affiliated with University of Nottingham and other places

Leishmaniasis is a debilitating and often fatal neglected tropical disease. Males from sub-populations of the Leishmania-harbouring sandfly, Lutzomyia longipalpis, produce the diterpene sex and aggregation pheromone, sobralene, for which geranylgeranyl diphosphate (GGPP) is the likely isoprenoid precursor. We have identified a GGPP synthase (lzGGPPS) from L. longipalpis, which was recombinantly expressed in bacteria and purified for functional and kinetic analysis. In vitro enzymatic assays using LC-MS showed that lzGGPPS is an active enzyme, capable of converting substrates dimethylallyl diphosphate (DMAPP), (E)-geranyl diphosphate (GPP), (E,E)-farnesyl diphosphate (FPP) with co-substrate isopentenyl diphosphate (IPP) into (E,E,E)-GGPP, while (Z,E)-FPP was also accepted with low efficacy. Comparison of metal cofactors for lzGGPPS highlighted Mg2+ as most efficient, giving increased GGPP output when compared against other divalent metal ions tested. In line with previously characterised GGPPS enzymes, GGPP acted as an inhibitor of lzGGPPS activity. The molecular weight in solution of lzGGPPS was determined to be ∼221 kDa by analytical SEC, suggesting a hexameric assembly, as seen in the human enzyme, and representing the first assessment of GGPPS quaternary structure in insects.

Background: High molecular weight kininogen (HK) circulates in plasma as a complex with zymogen prekallikrein (PK). HK is both substrate and co-factor for activated plasma kallikrein (PKa) and the principal exosite interactions occur between PK N-terminal apple domains and the C-terminal D6 domain of HK. Objective: To determine the structure of the complex formed between PK apple domains and a HKD6 fragment and compare this to the FXI-HK complex. Methods: We produced recombinant FXI and PK heavy chains (HC) spanning all four apple domains. We co-crystallised PKHC (and subsequently FXIHC) with a 31 amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analysed the full length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry (HDX-MS). Results and conclusions: The 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3. The alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Phage display (PD) is a powerful method and has been extensively used to generate monoclonal antibodies and identify epitopes, mimotopes, and protein interactions. More recently, the combination of next-generation sequencing (NGS) with PD (NGPD) has revolutionized the capabilities of the method by creating large data sets of sequences from affinity selection-based approaches (biopanning) otherwise challenging to obtain. NGPD can monitor motif enrichment, allow tracking of the selection process over consecutive rounds, and highlight unspecific binders. To tackle the wealth of data obtained, bioinformatics tools have been developed that allow for identifying specific binding sequences (binders) that can then be validated. Here, we provide a detailed account of the use of NGPD experiments to identify ubiquitin-specific protease peptide ligands.Key wordsPhage displayNext-generation sequencingBiopanningBioinformatical analysisDeubiquitinasePeptides

Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway. Post-translational modifications are critical for regulating the DNA damage response. Here, the authors identify a methylation-deubiquitination crosstalk between methyltransferase PRMT1 and deubiquitinase USP11, showing that the enzymes regulate each other’s functions in DNA repair.

Metabolism is highly interconnected and also has profound effects on other cellular processes. However, the interactions between metabolites and proteins that mediate this connectivity are frequently low affinity and difficult to discover, hampering our understanding of this important area of cellular biochemistry. Therefore, we developed the MIDAS platform, which can identify protein-metabolite interactions with great sensitivity. We analyzed 33 enzymes from central carbon metabolism and identified 830 protein-metabolite interactions that were mostly novel, but also included known regulators, substrates, products and their analogs. We validated previously unknown interactions, including two atomic-resolution structures of novel protein-metabolite complexes. We also found that both ATP and long-chain fatty acyl-CoAs inhibit lactate dehydrogenase A (LDHA), but not LDHB, at physiological concentrations in vitro. Treating cells with long-chain fatty acids caused a loss of pyruvate/lactate interconversion, but only in cells reliant on LDHA. We propose that these regulatory mechanisms are part of the metabolic connectivity that enables survival in an ever-changing nutrient environment, and that MIDAS enables a broader and deeper understanding of that network.

A careful balance between active-site and exosite contributions is critically important for the specificity of many proteases, but this balance is not yet defined for some of the serine proteases that serve as coagulation factors. Basavaraj and Krishnaswamy have closed an important gap in our knowledge of coagulation factor X activation by the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, which they describe as a “dock and lock.” This finding not only significantly enhances our understanding of this step in the coagulation cascade and highlights parallels with the prothrombinase complex, but will also provide a novel rationale for inhibitor development in the future.

The contact system is composed of Factor XII (FXII), prekallikrein (PK) and co-factor kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+ dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion with residues Arg36 and Arg65 forming contacts with two distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII binding site and a comparison with the ligand free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+ binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with SPR demonstrate the gC1qR Zn2+ site contributes to FXII binding and plasma based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only one high affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher order 500kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors which may be a prelude for initiating the cascades which drive bradykinin generation and the intrinsic pathway of coagulation.