Publication highlights

When lysosomes—the cell’s recycling centres—get damaged, several defence systems are activated to prevent cell death. One important repair process involves close contact between the lysosome and the endoplasmic reticulum. This process uses certain proteins and lipids, including PI4K2A, but how PI4K2A reaches damaged lysosomes was unknown. Researchers at the Crick found that vesicles containing the ATG9A protein are responsible for delivering PI4K2A to damaged lysosomes during injury or bacterial infection. Another protein, ARFIP2, also found in the ATG9A vesicles, helps control lipid levels on lysosomes and aids in recycling the vesicles, keeping lysosomes healthy after damage or infection.

Researchers from the Francis Crick Institute and the Gulbenkian Institute for Molecular Medicine (GIMM) have shown that the evolution of a family of exported proteins in the malaria-causing parasite Plasmodium falciparum enabled it to infect humans. The team looked at over two thousand P. falciparum samples from people infected with malaria, finding that out of 21 FIKK kinases, 18 were protected against harmful mutations, suggesting they are necessary for the parasite to infect humans and likely helped it evolve. The researchers then expressed the FIKK kinases in bacteria to see what each one does. This experiment showed that the FIKK kinases all had different protein targets in the cell. Finally, the team showed that the specificity of FIKK kinases is linked to small changes in a flexible loop region, and that two molecules could block most FIKK kinases in a test tube. Blocking all FIKK kinases could be a promising treatment strategy for malaria.

Toxoplasma is a single-cell parasite that infects any warm-blooded animal. It can persist for a long time in the host as it can withstand pathogen-clearing mechanisms. How the parasite circumvents clearance in a wide host range, with different immune mechanisms, remains unknown. To prevent being killed, the parasite secretes ~250 proteins into the host cell. Which of these effector proteins enable infection of all species, and in parasite strains that are particularly virulent in humans, has not been established. Researchers at the Crick and GIMM identified a core set of proteins required for survival in different mouse species with varying susceptibility to Toxoplasma infection. Deletion of the top hit, a protein called GRA12, led to increased host-cell death and early exit of the parasite from the infected cell. The team propose that instead of one virulence factor required across all species, the parasite evolved a suite of effector proteins to counter unique clearance mechanisms in different hosts.

The modification of proteins with a small regulatory protein called ubiquitin influences the majority of cellular functions and malfunction is implicated in many diseases. To capitalise on the therapeutic potential of regulating ubiquitination processes, we need to understand the mechanisms of the enzymes that catalyse it: E3 ubiquitin ligases. Researchers at the Crick characterise a previously unrecognised sub-family of ‘pseudoligases’, which lack key structural and catalytic features. These deviations mean that they cannot catalyse ubiquitination but instead appear to regulate active E3 ligases. Uncovering this unexpected evolutionary strategy takes us a step closer to understanding and manipulating the ubiquitin system.

Researchers at the Crick previously discovered that the tail of Influenza virus M2 (matrix 2) protein binds directly to the autophagy (self-eating) protein LC3, which becomes attached to membranes following collapse of pH gradients during infection. In this paper, the team describes a crystal structure of the M2 tail bound to LC3, and report that an unstructured region directly upstream of the interaction is a caspase cleavage motif. Caspases are proteases which can cleave cellular proteins during cell death. In this case, the paper shows that caspase cleavage of M2 disrupts the interaction between M2 and LC3. Functionally, this affects M2 transport to the plasma membrane for virion budding, also disrupts influenza from forming long filaments at the cell surface. This is speculated to be a mechanism to change the structure of virions during cell death, to one that does not require as many cellular resources.

Small molecule probes offer powerful tools for the study of biological systems and can serve as starting points for the development of therapeutics. The vast majority of human proteins lack such chemical tools, which hinders our ability to explore their function in the context of health and disease. Screening libraries of “reactive fragments”, small molecules that form covalent bonds with their protein targets, by mass spectrometry enables the discovery of new ligands in the native cellular environment. Together with GSK as part of the Crick-GSK Biomedical LinkLabs Prosperity Partnership, researchers at the Crick have developed a robust and versatile proteomics platform for profiling of cysteine-reactive fragments against the native proteome and have identified hundreds of new protein-ligand interactions for probe development.

Covalent drugs - which bind irreversibly to their targets - have increased potency and reduce the frequency a dose must be taken. However, it's challenging to design peptide inhibitors for enzymes, let alone to further alter them to contain a reactive group which will form a covalent bond to the enzyme. Researchers at the Crick used a specialised screening system called RaPID to identify irreversible, high affinity binders for a target of interest. This enabled them to go from a library of 1 trillion peptides down to an enriched library of peptides that tightly bind to a protein target. They incorporated unnatural amino acids with an irreversibly-binding 'warhead' into the peptide library, which enabled covalent binding to the target. The new system was used to identify several covalent peptides which tightly bind to the protein target PADI4, which is misregulated in rheumatoid arthritis, lupus and several cancers. These peptides, which also inhibit PADI4 activity, could form the basis of drugs for these diseases.

Microglia are important in maintaining the healthy brain but can contribute to nerve damage in amyotrophic lateral sclerosis (ALS) through largely unknown mechanisms. Researchers at the Crick studied microglia derived from human stem cells carrying ALS-causing mutations in the VCP gene. They compared ALS microglia to healthy microglia, before and after inducing inflammatory responses using a bacterial toxin called lipopolysaccharide (LPS). The VCP mutant microglia displayed different activation of inflammatory pathways compared to the healthy microglia. Mutant microglia also showed similar altered gene expression in a mouse model of ALS and postmortem tissue from people with sporadic ALS. VCP-mutant microglia also showed dysfunction independent of a gene called GPNMB, which was thought to play a role in ALS, and also induced specific responses in neighbouring nerve cells and another type of glia called astrocytes.