Research Highlights

New directions in Alzheimer research

Alzheimer’s Disease (AD) is a neurodegenerative disease, characterised by the deposition of amyloid b (Ab) in senile plaques. The correlation between senile plaques and loss of cognitive functions was first discovered in 1906. Research in the late 20th century revealed that the neurons affected are predominantly those secreting the neurotransmitter acetylcholine, and that the key enzyme terminating cholinergic signals, acetylcholinesterase (AChE), is commonly found in senile plaques. However, the relationship between Ab and AChE and their role in AD progression are not fully resolved.

One of the synaptic AChE variants has a unique oligomerisation domain, a portion of which is homologous to Ab and from which peptides can be generated by insulin-degrading enzyme (an amyloid scavenger). These peptides adopt an amyloid b-sheet conformation, form amyloid fibrils and enhance Ab amyloidogenesis. The Vaux group has demonstrated that these peptides in an amyloid b-sheet conformation occur naturally in an AD transgenic mouse model, colocalise with Ab at the core of senile plaques, and are involved in disease progression. This discovery may initiate new therapeutic strategies to combat AD.

Written by Shaked Ashkanazi

Létitia Jean, Stephen Brimijoin and David J. Vaux (2019).  In vivo localization of human acetylcholinesterase-derived species in a β-sheet conformation at the core of senile plaques in Al

JBC doi: 10.1074/jbc.RA118.006230

Keeping back the neutrophils – a new role for the Cannabinoid Receptor CB2 in inflammation

The cannabinoid receptors CB1 and CB2 were discovered over 30 years ago. While he CB1 receptor is expressed predominantly in the brain and is well known for mediating the psychoactive effects of the recreational drug marijuana, the CB2 cannabinoid receptor is expressed in the immune system, where its exact role has remained largely enigmatic.

Recent work from the Greaves lab in collaboration with Dunn School alumnus Asif Iqbal, now at the University of Birmingham, has shown that the CB2 receptor plays a non-redundant role in preventing excessive harmful inflammation. A series of experiments performed in CB2 knockout mice showed that signalling via CB2 restricts neutrophil mobilisation to sites of infection or injury, where they can exacerbate inflammation.  Experiments in mice were confirmed by pharmacological experiments performed using human neutrophils. This discovery identifies CB2 as a potential target for developing new treatments that might reduce inflammation in diseases such as rheumatoid arthritis and inflammatory bowel disease.

Written by Shaked Ashkanazi

Kapellos, T. S., Taylor, L., Feuerborn, A., Valaris, S., Hussain, M. T., Rainger, G. E., Greaves, D. R., Iqbal, A. J. (2019). Cannabinoid receptor 2 deficiency exacerbates inflammation and neutrophil recruitment.

The FASEB Journal

Signalling before the exit: a tight checkpoint at the mitotic spindle

Cell division requires highly-controlled events to ensure faithful separation of DNA. Many of the pivotal signals governing mitosis are kinases and phosphatases that activate/inactivate protein substrates via phosphorylation or dephosphorylation. Prior to mitotic exit, cells monitor the attachment of chromosomes, via the kinetochores, to the mitotic spindle using the spindle checkpoint, which can trigger cell cycle arrest when errors are detected.

Spindle checkpoint signalling is initiated by the recruitment of MPS1 kinase to unattached kinetochores, yet how the spindle checkpoint is kept responsive to late-stage spindle defects is unknown. A study by Hayward and colleagues from the Gruneberg lab, in collaboration with the Barr and Novak labs (Biochemistry), has revealed the mechanism by which cells restrict MPS1 localisation to a defined time during mitosis. In particular, they show that CDK1-CyclinB1 promotes MPS1 recruitment and is opposed by the phosphatase PP2A-B55, which terminates signalling before mitotic exit. They nailed down the key residue in MPS1’s kinetochore binding domain (Ser281) targeted by this regulatory mechanism that allows cells to localise MPS1 and sustain the checkpoint-responsive window until chromosome segregation but not beyond, thus avoiding fatal cell division errors.

Written by Anna Caballe @caballe_anna

Hayward D, Alfonso-Pérez T, Cundell MJ, Hopkins m, Holder J, Bancroft J, Hutter LH, Novak B, Barr FA, Gruneberg U. (2019). CDK1-CCNB1 creates a spindle checkpoint permissive state by enabling MPS1 kinetochore localisation

J Cell Biol DOI: 10.1083/jcb.201808014 |

Cell division throttle is the brakes as well: CDK1 is also a spindle checkpoint protein

Cell division is regulated by the protein Cyclin B-dependent kinase (CDK1-CCNB1). A master cell cycle regulator, CDK1 promotes entry into mitosis, and also activates the proteins that prevent mitotic exit. These proteins, such as MPS1 and MAD1, are known as spindle checkpoint proteins. They function by localising to unattached kinetochores, preventing the completion of mitosis until the kinetochores of all chromosomes are properly attached to the spindle apparatus through microtubules. This ensures that daughter cells are free from errors in chromosome number.

In a joint study, Daniel Hayward of Ulrike Gruneberg’s lab and Tatiana Alfonso-Pérez of Francis Barr’s lab (Biochemistry) and colleagues show that in addition to promoting mitosis, CDK1-CCNB1/CycB1 surprisingly functions as a bona fide spindle checkpoint protein. They demonstrate that CCNB1 (CycB1) is recruited to unattached kinetochores by a direct interaction with the spindle checkpoint protein MAD1. They additionally show that CCNB1 is necessary for a positive-feedback loop that recruits the key spindle checkpoint regulator MPS1 in a timely manner upon mitotic entry and sustains spindle checkpoint arrest (see RH on Hayward et al. 2019 for further details).

Written by Derek Xu @derekcxu

Alfonso-Pérez T, Hayward D, Holder J, Gruneberg U, Barr FA. (2019).   MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling

J Cell. Biol. DOI: 10.1083/jcb.201808015 

RH31_Rack et al_v2.png

Protein Data Bank Europe

Lessons from ancient fish on the selectivity and inhibition of protein modifications

ADP-ribosylation is a post-translational modification characterised by the addition of ADP-ribose on target proteins, a modification that is placed on a variety of residues. The specific nature of the modification determines the effect on many important processes, including DNA damage responses and the regulation of programmed cell death.

In vertebrates, two of the enzymes that remove ADP-ribose are ARH1 (cleaves it from arginine residues) and ARH3 (from serine residues). Rack and colleagues from Ivan Ahel’s lab set out to investigate the underlying differences between these enzymes. To that end, they solved the 3D structures of the ARH1/3 enzymes and their substrates to atomic level. Since human ARH3 was reluctant to crystalise, they scanned known genomes of alternative species and found an ancient orthologue: coelacanth (Latimeria chalumnae), a “living fossil” that has barely changed in 400 million years. The resulting structures revealed the structural basis for the differences. Together with supporting experiments, they represent an important step in our understanding of how these enzymes select their targets. This will be useful in designing selective drugs to target these essential enzymes

Written by Shaked Ashkanazi

Rack JGM, Ariza A, Drown BS, Henfrey C, Bartlett E, Shirai T, Hergenrother PJ, Ahel I (2018). (ADP-ribosyl)hydrolases: Structural Basis for Differential Substrate Recognition and Inhibition

Cell Chem. Biol. 25(12):1533-1546.e12. doi: 10.1016/j.chembiol.2018.11.001. Epub 2018 Nov 21.

Consequences of excessive noncoding transcription unveiled.

Only a fraction of the human genome codes for protein-coding genes, yet these are the most studied regions. Other elements of the genome code for non-coding RNA but the regulation and functions of these other genes remains largely unknown. Transcription, the conversion of DNA to RNA, occurs across both protein-coding and RNA-coding genes.

Takayuki Nojima, Michael Tellier et al, from the Proudfoot and Murphy labs, respectively, have shown that a transcription-associated elongation factor, SPT6 plays a role in maintaining the balance of transcription between these two types of genes by modifying the chromatin. This factor can also affect the overall transcription level of long non-coding RNA genes. Higher non-coding RNA transcription can be associated with DNA damage, through the formation of R-loops, and uncontrolled transcription of non-coding RNAs can clash with the DNA replication apparatus promoting cellular senescence. This detailed molecular study highlights the importance for the cell to control the expression of non-coding regions of the genome and indicates the presence of a molecular mechanism dependent on SPT6 differentiating transcription of protein-coding and long non-coding RNA genes.

Written by Heather Jeffery @HeatherMJeffery

Nojima T, Tellier M, Foxwell J, Ribeiro de Almeida C, Tan-Wong SM, Dhir S, Dujardin G, Dhir A, Murphy S, Proudfoot NJ. (2018) Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senes

Mol Cell. pii: S1097-2765(18)30843-8. doi: 10.1016/j.molcel.2018.10.011. 

New tools to explore the question of life or death in caspase-activating cells

Caspases are protein-cleaving enzymes best known as master regulators of programmed cell death through apoptosis. Sequential cleavage steps firstly activate initiator caspases and then downstream effector caspases, to dismantle all of the cell components during apoptosis. Unexpectedly, recent investigations also implicate this protein family in non-apoptotic functions (for example cell proliferation, differentiation, and migration). Although many sensors have successfully shown effector caspase activation in vivo, few tools are available to directly monitor initiator caspase activity. This is key to fully understand caspase functions.

Alberto Baena-Lopez and his group have developed a suitable sensor to visualise initiator caspase activation in Drosophila tissues. Live imaging of these sensors has revealed unknown events of apoptosis that precede the previously described stages of apoptosis in flies. Strikingly, they are also able to show stereotyped patterns of caspase activation unlinked to apoptosis in many Drosophila tissues. Their novel system reports on caspase function at both a cellular and organismal level, and it promises to provide further insight into the biology of these proteins in healthy and apoptotic cells.

Written by Zoe Geraghty @zoe_geraghty

Baena-Lopez LA, Arthurton L, Bischoff M, Vincent JP, Alexandre C, McGregor R (2018) Novel initiator caspase reporters uncover unknown features of caspase-activating cells.

Development dev.170811

Secreting secrets no more

Protein secretion systems are essential for many bacteria; they play roles in communication, virulence factor delivery, protection against harmful agents and nutrient uptake. A certain group of pathogenic Gram-negative bacteria use the Type 9 secretion system (T9SS), a protein complex that spans the cell envelope. The T9SS is key to bacterial motility and is an essential determinant of pathogenicity in severe periodontal disease. However, the identity and structure of the central transport channel (translocon) was unclear – until now.

Using different biochemical and structural approaches, Lauber, Deme (Lea lab) and colleagues identified SprA as the translocon.  The structure of SprA, solved by cryo-electron microscopy, forms an unprecedented 36-stranded β-barrel in the outer membrane – the largest observed to date. Additionally, SprA forms complexes with three other accessory proteins (PPI, Plug and PorV), which control access to the SprA barrel. Taken together, this research has uncovered the structure and function of a critical part of the protein translocation T9SS machinery, which is directly involved in the transport of virulence factors in disease-causing bacteria.

Written by Lisa Gartenmann

Lauber F, Deme JC, Lea SM, Berks BC. Type 9 secretion system structures reveal a new protein transport mechanism.

Nature. 2018 Nov 7. doi: 10.1038/s41586-018-0693-y.

Putting the pieces together: new insights into RNA splicing

Eukaryotic genes are transcribed by the RNA polymerase II (Pol II) into mRNAs, which are then translated into protein products. These genes also contain non-coding stretches of DNA called introns; these are transcribed into RNA, but must be cut out before the final functional protein is produced. To ensure this, a multi-molecular complex called the spliceosome assembles on RNAs as they are being transcribed and splices the coding regions (exons) directly together, removing the introns. How two distant sites are physically brought together to be spliced has remained unknown.

Taka Nojima (Proudfoot group) and colleagues at the University of Lisbon have previously developed a technique to sequence elongating transcripts (mNET-seq). In a recent study, they couple this with mass spectrometry to show that the active spliceosome associates with Pol II, physically tethering the cleaved upstream exon in position while transcription continues. Interestingly, the association is specific to a Pol II modification (S5P CTD) previously thought to act only in transcription initiation. They also extensively characterise co-transcriptional splicing intermediates, providing significant insight into the mechanism and coordination of mammalian co-transcriptional splicing.

Written by Zoe Geraghty @zoe_geraghty

Nojima T, Rebelo K, Gomes T, Grosso AR, Proudfoot NJ, Carmo-Fonseca M (2018). RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing

Mol. Cell 72(2):369-379.e4.

New Rules for the Histone Modification Code

Post-translational protein modifications (PTMs) are important for the regulation of many cellular processes. Of these, ADP-ribosylation (ADPr)—the reversible addition of ADP-ribose molecules—of histones can be particularly important for DNA damage response. Previous work in Ivan Ahel’s lab found that ADPr on serine residues (Ser-ADPr) is the most prominent type of ADPr following DNA damage. Prior to this, the exact location of ADPr on many proteins, including histones, remained elusive. Furthermore, the study of ADPr and other histone PTMs had never intersected before, despite how different combinations of histone PTMs act together in a code to regulate important nuclear functions.

In this study, Bartlett et al., examined the interplay of histone marks, and introduced a simple method to detect Ser-ADPr peptides, overcoming limitations on currently available techniques. The study reveals how Ser-ADPr is mutually exclusive with proximal phosphorylation and acetylation—other PTMs—at N-terminal tails of histones. These results indicate that the context of the local histone code affects Ser-ADPr, and vice versa. Additionally, they discovered that tyrosine residues can also undergo ADPr, whose physiological relevance is still unclear.

Written by Derek Xu @derekcxu

Bartlett E, Bonfiglio JJ, Prokhorova E, Colby T, Zobel F, Ahel I, Matic I. (2018). Interplay of Histone Marks with Serine ADP-Ribosylation.

Cell Rep. 24(13):3488-3502.e5. doi: 10.1016/j.celrep.2018.08.092.