Research Highlights

Tracking the fate of orphan proteins

While some proteins in the cell function on their own, most have to assemble into multiprotein complexes to perform their function. Subunits failing to integrate into a complex, may become toxic and must be degraded. It is not clear how the cell identifies these “orphan” subunits and distinguishes them from newly made subunits which are yet to be utilized. While investigating this process in the context of protein complexes destined for endoplasmic reticulum (ER) membrane, Dr. Nivedita Natarajan and her colleagues from Prof. Carvalho group made an unexpected observation: many unassembled subunits were degraded only in the inner nuclear membrane, a highly specialized region of the ER.  

The ER membrane is continuous with the inner nuclear membrane (INM), however, the functions and protein content of ER and INM are quite different. In their previous work, the Carvalho laboratory showed that ER proteins which erroneously diffused into the INM are recognised and degraded by the Asi protein complex. Dr. Natarajan and colleagues found that the same complex is responsible for degradation of orphan/unassembled subunits of ER localized complexes. The authors concluded that restriction of quality control of unassembled subunits to the INM provides a mechanism protecting the complex subunits from premature degradation. Their work was developed using baker’s yeast as a model system. In the future, it will be interesting to investigate whether spatial restricted quality control processes operate in higher eukaryotes, like humans.

Written by Lucie Kafkova

Natarajan N, Foresti O, Wendrich K, Stein A, Carvalho P (2019).
Quality Control of Protein Complex Assembly by a Transmembrane Recognition Factor.

Mol Cell. pii: S1097-2765(19)30763-4

Positive feedback loop prepares cells for division

Cell division is a multi-stage process, which involves dividing the DNA into two daughter cells. Centrosomes organise microtubules, which help distribute chromosomes during cell division, as well as provide the cell with a structure. Centrosomes are made of two cylinders (called centrioles) wrapped in a pericentriolar material (PCM) which grows as the cell gets ready for division. The components that make up this PCM are conserved between most animals, thus working with Drosophila (fruit flies) can provide insights relevant to a variety of organisms.

Alvarez-Rodrigo et al, from the Raff lab, have studied the recruitment and subsequent organisation of some of the key components of the PCM in flies (Spd-2, Polo and Cnn). By making flies carrying mutant forms of Spd-2 that cannot interact with Polo they have discovered that these 3 key proteins have to co-operate in order for the PCM to grow in size in fly embryos. Identification of these interactions gives us a better understanding of the requirements for a properly functioning centrosome, thus developing our overall knowledge of the process of cell division, a process that is constantly happening within our bodies.

Written by Heather Jeffery @HeatherMJeffery

Alvarez-Rodrigo I, Steinacker TL, Saurya S, Conduit PT, Baumbach J, Novak ZA, Aydogan MG, Wainman A, Raff JW. 
Evidence that a positive feedback loop drives centrosome maturation in fly embryos.

eLife 2019;8:e50130

MPS1 kinase regulation by PP2A-B56 is vital for successful mitosis

The ultimate goal of mitosis is to produce two daughter cells with the same genetic make-up as the parent cell. It consists of several steps, kept in check by proteins such as kinases and phosphates and deregulation of which is associated with human pathologies such as cancer.

One important kinase in this process is Mono-Polar Spindle 1 (MPS1), which monitors the correct formation of microtubule-kinetochore attachments and initiates spindle assembly checkpoint signalling in case of errors. MPS1 is also actively involved in resolving incorrect kinetochore attachments by phosphorylating outer kinetochore proteins. Accurate regulation of MPS1 kinase activity, via autophosphorylation of the MPS1 T-loop, is therefore critical for faithful chromosome segregation.

Hayward and colleagues from the Gruneberg lab have now shown that a kinetochore-associated pool of the PP2A-B56 phosphatase regulates the T-loop autophosphorylation of MPS1 and hence its kinase activity. Expression of a constitutively active form of MPS1 refractory to PP2A-B56 dephosphorylation results in exaggerated MPS1-mediated error correction, mitotic delays and impaired cell cycle progression, stressing the importance of balanced kinase and phosphatase activities for successful mitosis.

Written by Iqbal Dulloo

Hayward D, Bancroft J, Mangat D, Alfonso-Pérez T, Dugdale S, McCarthy J, Barr FA, Gruneberg U. (2019)
Checkpoint signalling and error correction require regulation of the MPS1 T-loop by PP2A-B56.

http://doi.org/10.1083/jcb.201905026

First structures of the genome replication machinery of influenza A virus

Influenza A viruses are the most common cause of seasonal flu in humans and also infect animals, representing significant public health and economic burdens. During infection, the virus invades host cells and makes many copies of its RNA genome to produce new virus particles. RNA-dependent RNA polymerase is the enzyme responsible for this genome replication.

A collaboration between the Fodor group and the group of Jonathan Grimes (Division of Structural Biology, University of Oxford) published in Nature has revealed how influenza A virus RNA polymerase replicates the genome. Using cryo-electron microscopy and X-ray crystallography, they have produced the first high-resolution structures of human and avian influenza A virus RNA polymerases. Their results show that the influenza A virus RNA polymerase forms a dimeric complex, where one of the polymerases activates the other to copy RNA. However, if dimerisation is blocked viral genome replication is inhibited, meaning the virus cannot propagate. Therefore, the dimerisation interface is an attractive target for antiviral drug development, made possible by these first structures.

 

Written by Isabella Maudlin

Fan H, Walker AP, Carrique L, Keown JR, Serna Martin I, Karia D, Sharps J, Hengrung N, Pardon E, Steyaert J, Grimes JM, Fodor E (2019).
Influenza A virus RNA polymerase structures provide insights into viral genome replication

https://doi.org/10.1038/s41586-019-1530-7

Neisseria: friend or foe

The bacterial genus Neisseria comprises commensal and pathogenic species. Pathogenic Neisseria can be found in human nasal passages where the bacteria attach to the surface using special filaments, called Type IV pili. These are required both during harmless colonisation of the upper airway, and invasive disease.

Mariya Lobanovska and colleagues studied Neisseria meningitidis, a pathogenic member of the Neisseria species, which causes meningitis and septic shock. They studied the components of the Type IV pili and found that one version of a component called PilE is particularly common in strains which cause epidemic disease. Interestingly, this version is conserved and shares homology with PilE from commensal Neisseria. To understand how this version can be present in both pathogenic and commensal bacteria, the transcriptional regulation was investigated. This new paper shows that the transcriptional regulation differs between the pathogenic and commensal bacteria. This new understanding of PilE transcription provides insight into the divergent mechanisms of Type IV pili regulation in commensal and pathogenic Neisseria.

 

Written by Heather Jeffery @HeatherMJeffery

Lobanovska M, Tang CM, Exley RM.
The contribution of σ70 and σN factors to expression of class II pilE in Neisseria meningitidis

J. Bacteriol pii: JB.00170-19. doi: 10.1128/JB.00170-19. [Epub ahead of print]

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Figure legend: To study parasite swimming behaviour in vitro, the swimming speed and directionality of 100 Leishmania knockout lines was measured individually (data plot). A selection of these mutant lines (coloured symbols) was passaged though sand flies

Leishmania’s tail crucial for sand fly colonisation

Leishmaniais a parasite that causes leishmaniasis, a disease that affects millions of people in Mexico and Central America. The parasite is passed between mammals by the bite of sand flies. Leishmaniahas a flagellum, a long tail-like organelle primarily used for direction and speed of motion, that is remodelled depending on the phase of its life cycle.

Tom Beneke and his colleagues (Gluenz Lab) systematically studied the function of flagellar proteins of Leishmania mexicana by using proteomics and CRISPR gene editing technologies. They first identified the proteins that constitute Leishmania’s flagellum by mass spectrometry. With this knowledge, they generated a library of 100 knockout mutants, each of which lacked a flagellar protein. They showed that many mutants swam slower than normal Leishmania,while some could swim faster; the rest were completely immobilised. They later showed that two mutants, one immobile (PF16 knockdown) and one uncoordinated swimmer (MBO2 knockdown), failed to fully colonise sand flies. This comprehensive study sheds light on the importance of directional swimming mediated by flagellum for Leishmaniainfection of sand flies, which in turn infect humans.

Written by Sheng Kai Pong

Beneke T, Demay F, Hookway E, Ashman N, Jeffery H, Smith J, Valli J, Becvar T, Myskova J, Lestinova T, Shafiq S, Sadlova J, Volf P, Wheeler RJ, Gluenz E. Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections. PLoS Pathog. 2019 Jun 26;15(6):e1007828. doi: 10.1371/journal.ppat.100782



Conservation of bacterial protein export system

Certain bacteria use a protein complex called the injectisome to deliver proteins into host cells, effectively increasing their pathogenic potential. The injectisome “needle” grows from a protein scaffold around its base called the basal body, which is related to the basal body of the bacterial flagellum. Bacterial proteins destined for export are picked up by an export apparatus complex (EA) inside the basal body. EA then exports the proteins across bacterial envelope into the flagellum or injectisome. In the flagellum, three of the EA proteins form an export gate core complex (FliPQR) and assemble into a helical structure that forms the start of the channel which culminates in the flagellum.

Research led by Steven Johnson and Lucas Kuhlen from Susan Lea’s lab was aimed at investigating the structure of the core export gate complex in the bacterial injectisome (SctRST). Single-particle cryo-electron microscopy of the export gate purified from human pathogen Shigella revealed a structure with striking similarity to the flagellar export complex gate. This finding further solidifies the conservation of the apparatus supporting the function of bacterial flagella and injectisomes.

Written by Lucie Kafkova

Johnson S, Kuhlen L, Deme JC, Abrusci P, Lea SM.
The Structure of an Injectisome Export Gate Demonstrates Conservation of Architecture in the Core Export Gate between Flagellar

mBio 10 (3) e00818-19; DOI: 10.1128/mBio.00818-19

RNA as a target for ADP-ribosylation

The human genome encodes several poly(ADP-ribose) polymerases (PARPs), which are enzymes involved in ADP-ribosylation, a reversible chemical modification, of macromolecules such as proteins and DNA. ADP-ribosylation occurs in both prokaryotes and eukaryotes and is linked to vital cellular processes namely stress responses, DNA repair, host-virus interactions, etc. This modification is mostly associated with proteins and to some extent, DNA. It was recently proposed that RNA molecules could also be cellular targets of ADP-ribosylation.

Deeksha Munnur and colleagues from Ivan Ahel’s lab, have now shown that ADP-ribosylation of RNA molecules indeed happens and is a more common phenomenon than previously believed. They found that ADP-ribosylation occurs at the terminal phosphate of RNA forming a non-canonical RNA cap and can be regulated by both PARP-like proteins from bacteria and by some human PARPs. Furthermore, they provide the first evidence that ADP-ribosylation of RNA is a reversible process that can be controlled by ADP-ribosylhydrolases from human as well as VEEV and SARS viruses. This study shines a light on a new regulatory mechanism of RNA molecules with potential therapeutic relevance.

Written by Iqbal Dulloo

Munnur D, Bartlett E, Mikolčević P, Kirby IT, Matthias Rack JG, Mikoč A, Cohen MS, Ahel I.
Reversible ADP-ribosylation of RNA.

Nucleic Acids Res. 47(11): 5658-5669

Understanding Pandemic Flu

Influenza A viruses are responsible for both seasonal outbreaks of respiratory disease and occasional pandemics. Their genome is made of eight RNA segments. If two different virus strains of infect the same cell, they can “shuffle” their segments instead of packaging just their own, and generate a new strain. This can result in the generation of a pandemic, since people may lack the immunity necessary to defeat the new virus. This happened in 1957 and 1968, when pandemic viruses killed millions worldwide. While we can detect this genomic “shuffling”, little is known about how they occur. Therefore, predicting them is still a challenge.

Research led by David Bauer in Ervin Fodor’s group addressed the question of the structure of the viral RNA genome. Collaborating with groups in Australia (Lorena Brown) and in the US (Alain Laederach), they employed high-throughput analysis techniques to reveal specific interactions that control packaging of RNA segments. Rarely, these interactions facilitate inter-strain “shuffling”. This work sheds light on influenza virus biology and is a major step in our understanding of influenza pandemic generation.

Written by Shaked Ashkanazi

Dadonaite B, Gilbertson B, Knight ML, Trifkovic S, Rockman S, Laederach A, Brown LE, Fodor E, Bauer DLV.
The structure of the influenza A virus genome.

Nature Microbiology (2019).

The role of sugars in bacterial adhesion

Bacteria display molecules on their outside to facilitate adhesion and interaction with surfaces. This is particularly important during the initial stages of infection and biofilm formation for pathogenic bacteria. This adhesive ability allows bacteria to move on surfaces, both living and non-living, and eventually adhere irreversibly to form biofilms.

The Bharat lab uses a commonly studied bacterium, Caulobacter crescentus, to investigate this adhesion process. There are two main methods of adhesion, via proteins or polysaccharides (sugars). The focus of this paper is polysaccharide-mediated adhesion, in which a specialized sugar called holdfast plays a key role. In this paper, Sulkowski et al use a range of structural biology techniques, including cryo-electron microscopy, to identify the proteins which anchor and display holdfast on the cell surface. By looking at the mechanism of positioning holdfast at the membrane and maintenance of its cellular position, the role of sugars in bacterial adhesion can be better understood.

Written by Heather Jeffery @HeatherMJeffery

Sulkowski NI, Hardy GG, Brun YV, Bharat TAM
A multi-protein complex anchors adhesive holdfast at the outer membrane of Caulobacter crescentus.

J. Bacteriol pii: JB.00112-19. doi: 10.1128/JB.00112-19. [Epub ahead of print]

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