Publications by authors named "Helen R Flynn"

20 Publications

  • Page 1 of 1

TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria.

EMBO J 2021 Apr 21:e106188. Epub 2021 Apr 21.

The Francis Crick Institute, London, UK.

Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.
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http://dx.doi.org/10.15252/embj.2020106188DOI Listing
April 2021

Ca signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Sci Adv 2021 Mar 24;7(13). Epub 2021 Mar 24.

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.
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http://dx.doi.org/10.1126/sciadv.abe5396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990342PMC
March 2021

Author Correction: YAP1/TAZ drives ependymoma-like tumour formation in mice.

Nat Commun 2020 Sep 28;11(1):4934. Epub 2020 Sep 28.

Kinases and Brain Development Laboratory, The Francis Crick Institute, London, NW1 1AT, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-18851-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522079PMC
September 2020

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme.

Elife 2020 09 25;9. Epub 2020 Sep 25.

Signalling and Transcription Laboratory, The Francis Crick Institute, London, United Kingdom.

PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.
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http://dx.doi.org/10.7554/eLife.61509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599070PMC
September 2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring.

Mol Cell 2020 09 4;79(6):917-933.e9. Epub 2020 Aug 4.

Chromosome Segregation Laboratory, The Francis Crick Institute, London NW1 1AT, UK. Electronic address:

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
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http://dx.doi.org/10.1016/j.molcel.2020.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507959PMC
September 2020

Assessing Budding Yeast Phosphoproteome Dynamics in a Time-Resolved Manner using TMT10plex Mass Tag Labeling.

STAR Protoc 2020 Jun 19;1(1):100022. Epub 2020 Jun 19.

Chromosome Segregation Laboratory, The Francis Crick Institute, London NW1 1AT, UK.

Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling permits multiplexed protein identification and quantitative analysis by tandem mass spectrometry (MS/MS). We have used this technology to label 20 samples collected in a time-resolved manner from a wild-type and phosphatase mutant background to characterize phosphoproteome dynamics. Here, we provide a detailed protocol for biological and mass spectrometry sample preparation and analysis. For complete details on the use and execution of this protocol, please refer to Touati et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2020.100022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357674PMC
June 2020

YAP1/TAZ drives ependymoma-like tumour formation in mice.

Nat Commun 2020 05 13;11(1):2380. Epub 2020 May 13.

Kinases and Brain Development Laboratory, The Francis Crick Institute, London, NW1 1AT, UK.

YAP1 gene fusions have been observed in a subset of paediatric ependymomas. Here we show that, ectopic expression of active nuclear YAP1 (nlsYAP5SA) in ventricular zone neural progenitor cells using conditionally-induced NEX/NeuroD6-Cre is sufficient to drive brain tumour formation in mice. Neuronal differentiation is inhibited in the hippocampus. Deletion of YAP1's negative regulators LATS1 and LATS2 kinases in NEX-Cre lineage in double conditional knockout mice also generates similar tumours, which are rescued by deletion of YAP1 and its paralog TAZ. YAP1/TAZ-induced mouse tumours display molecular and ultrastructural characteristics of human ependymoma. RNA sequencing and quantitative proteomics of mouse tumours demonstrate similarities to YAP1-fusion induced supratentorial ependymoma. Finally, we find that transcriptional cofactor HOPX is upregulated in mouse models and in human YAP1-fusion induced ependymoma, supporting their similarity. Our results show that uncontrolled YAP1/TAZ activity in neuronal precursor cells leads to ependymoma-like tumours in mice.
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http://dx.doi.org/10.1038/s41467-020-16167-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220953PMC
May 2020

A malaria parasite subtilisin propeptide-like protein is a potent inhibitor of the egress protease SUB1.

Biochem J 2020 01;477(2):525-540

Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, U.K.

Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite.
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http://dx.doi.org/10.1042/BCJ20190918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993865PMC
January 2020

CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle.

Nature 2019 11 13;575(7783):523-527. Epub 2019 Nov 13.

The Francis Crick Institute, London, UK.

The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
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http://dx.doi.org/10.1038/s41586-019-1744-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874499PMC
November 2019

Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery.

PLoS Biol 2019 05 10;17(5):e3000264. Epub 2019 May 10.

Faculty of Infectious Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom.

Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACβ) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.
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http://dx.doi.org/10.1371/journal.pbio.3000264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530879PMC
May 2019

Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.

EMBO J 2018 12 28;37(24). Epub 2018 Sep 28.

Kinases and Brain Development Laboratory, The Francis Crick Institute, London, UK

Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder.
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http://dx.doi.org/10.15252/embj.201899763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293278PMC
December 2018

Quantitative Phosphoproteomics Reveals the Signaling Dynamics of Cell-Cycle Kinases in the Fission Yeast Schizosaccharomyces pombe.

Cell Rep 2018 07;24(2):503-514

Cell Cycle Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Laboratory of Yeast Genetics and Cell Biology, Rockefeller University, New York, NY 10065, USA.

Multiple protein kinases regulate cell-cycle progression, of which the cyclin-dependent kinases (CDKs) are thought to act as upstream master regulators. We have used quantitative phosphoproteomics to analyze the fission yeast cell cycle at sufficiently high temporal resolution to distinguish fine-grain differences in substrate phosphorylation dynamics on a proteome-wide scale. This dataset provides a useful resource for investigating the regulatory dynamics of cell-cycle kinases and their substrates. For example, our analysis indicates that the substrates of different mitotic kinases (CDK, NIMA-related, Polo-like, and Aurora) are phosphorylated in sequential, kinase-specific waves during mitosis. Phosphoproteomics analysis after chemical-genetic manipulation of CDK activity suggests that the timing of these waves is established by the differential dependency of the downstream kinases on upstream CDK. We have also examined the temporal organization of phosphorylation during G1/S, as well as the coordination between the NDR-related kinase Orb6, which controls polarized growth, and other cell-cycle kinases.
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http://dx.doi.org/10.1016/j.celrep.2018.06.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057490PMC
July 2018

Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

Mol Cell 2018 05 10;70(4):707-721.e7. Epub 2018 May 10.

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4 mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4p53 mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.
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http://dx.doi.org/10.1016/j.molcel.2018.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972231PMC
May 2018

CDK Substrate Phosphorylation and Ordering the Cell Cycle.

Cell 2016 Dec;167(7):1750-1761.e16

Cell Cycle Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Laboratory of Yeast Genetics and Cell Biology, Rockefeller University, New York, NY 10065, USA.

S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes. However, it has been suggested that changes in the total level of CDK kinase activity, rather than substrate specificity, drive the temporal ordering of S phase and mitosis. Here, we present a phosphoproteomics-based systems analysis of CDK substrates in fission yeast and demonstrate that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK. This is achieved by rising CDK activity and the differential sensitivity of substrates to CDK activity over a wide dynamic range. This is combined with rapid phosphorylation turnover to generate clearly resolved substrate-specific activity thresholds, which in turn ensures the appropriate ordering of downstream cell-cycle events. Comparative analysis with wild-type cells expressing multiple cyclin-CDK complexes reveals how cyclin-substrate specificity works alongside activity thresholds to fine-tune the patterns of substrate phosphorylation.
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http://dx.doi.org/10.1016/j.cell.2016.11.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161751PMC
December 2016

TLR and TNF-R1 activation of the MKK3/MKK6-p38α axis in macrophages is mediated by TPL-2 kinase.

Biochem J 2016 09 11;473(18):2845-61. Epub 2016 Jul 11.

The Francis Crick Institute, Immune Signalling Laboratory, Mill Hill Laboratory, London NW7 1AA, U.K.

Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-β-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8(D270A/D270A) mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8(D270A) mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.
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http://dx.doi.org/10.1042/BCJ20160502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095906PMC
September 2016

Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity.

Elife 2016 06 15;5. Epub 2016 Jun 15.

Signaling and Transcription Group, Francis Crick Institute, London, United Kingdom.

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A.
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http://dx.doi.org/10.7554/eLife.15460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963197PMC
June 2016

Juxtamembrane shedding of Plasmodium falciparum AMA1 is sequence independent and essential, and helps evade invasion-inhibitory antibodies.

PLoS Pathog 2011 Dec 15;7(12):e1002448. Epub 2011 Dec 15.

Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.

The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However - in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins.
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http://dx.doi.org/10.1371/journal.ppat.1002448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3240622PMC
December 2011

Global identification of multiple substrates for Plasmodium falciparum SUB1, an essential malarial processing protease.

Infect Immun 2011 Mar 10;79(3):1086-97. Epub 2011 Jan 10.

Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important merozoite surface and PV proteins. These include the essential merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptry-associated protein 1) and the merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.
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http://dx.doi.org/10.1128/IAI.00902-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067499PMC
March 2011

Identification of Holliday junction resolvases from humans and yeast.

Nature 2008 Nov;456(7220):357-61

Genetic Recombination Laboratory, Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK.

Four-way DNA intermediates, also known as Holliday junctions, are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. Here we identify nucleases from Saccharomyces cerevisiae and human cells that promote Holliday junction resolution, in a manner analogous to that shown by the Escherichia coli Holliday junction resolvase RuvC. The human Holliday junction resolvase, GEN1, and its yeast orthologue, Yen1, were independently identified using two distinct experimental approaches: GEN1 was identified by mass spectrometry following extensive fractionation of HeLa cell-free extracts, whereas Yen1 was detected by screening a yeast gene fusion library for nucleases capable of Holliday junction resolution. The eukaryotic Holliday junction resolvases represent a new subclass of the Rad2/XPG family of nucleases. Recombinant GEN1 and Yen1 resolve Holliday junctions by the introduction of symmetrically related cuts across the junction point, to produce nicked duplex products in which the nicks can be readily ligated.
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http://dx.doi.org/10.1038/nature07470DOI Listing
November 2008

Protein expression by a Beijing strain differs from that of another clinical isolate and Mycobacterium tuberculosis H37Rv.

Microbiology (Reading) 2005 Apr;151(Pt 4):1139-1150

MRC Centre for Molecular and Cellular Biology, Department of Medical Biochemistry, University of Stellenbosch Medical School, PO Box 19063, Tygerberg, 7505, South Africa.

The Beijing strain family has often been associated with tuberculosis (TB) outbreaks and drug resistance worldwide. In this study the authors have compared the protein expression and antigen recognition profiles of a local Beijing strain with a less prevalent clinical isolate belonging to the family 23 strain lineage, and the laboratory strain Mycobacterium tuberculosis H37Rv. Using two-dimensional electrophoresis, liquid chromatography tandem mass spectrometry and Western blot analysis several proteins were identified as quantitatively increased or decreased in both clinical strains compared to H37Rv. Remarkably, the Beijing strain showed increased expression of alpha-crystallin and decreased expression of Hsp65, PstS1, and the 47 kDa protein compared to the other clinical strain and H37Rv. One- and two-dimensional Western blot analysis of antigens expressed by the three strains, using plasma from TB patients, confirmed differential antigen expression by strains and patient-to-patient variation in humoral immunity. These observed protein differences could aid the elucidation of mechanisms underlying the success of the Beijing strain family, measured by global dissemination, compared to other M. tuberculosis strains.
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http://dx.doi.org/10.1099/mic.0.27518-0DOI Listing
April 2005