Allosteric modulation of the GTPase activity of a bacterial LRRK2 homolog by conformation-specific Nanobodies

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer–monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer–monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations

LRRK2 Biology from structure to dysfunction: research progresses, but the themes remain the same

Since the discovery of leucine-rich repeat kinase 2 (LRRK2) as a protein that is likely central to the aetiology of Parkinson's disease, a considerable amount of work has gone into uncovering its basic cellular function. This effort has led to the implication of LRRK2 in a bewildering range of cell biological processes and pathways, and probable roles in a number of seemingly unrelated medical conditions. In this review we summarise current knowledge of the basic biochemistry and cellular function of LRRK2. Topics covered include the identification of phosphorylation substrates of LRRK2 kinase activity, in particular Rab proteins, and advances in understanding the activation of LRRK2 kinase activity via dimerisation and association with membranes, especially via interaction with Rab29. We also discuss biochemical studies that shed light on the complex LRRK2 GTPase activity, evidence of roles for LRRK2 in a range of cell signalling pathways that are likely cell type specific, and studies linking LRRK2 to the cell biology of organelles. The latter includes the involvement of LRRK2 in autophagy, endocytosis, and processes at the trans-Golgi network, the endoplasmic reticulum and also key microtubule-based cellular structures. We further propose a mechanism linking LRRK2 dimerisation, GTPase function and membrane recruitment with LRRK2 kinase activation by Rab29. Together these data paint a picture of a research field that in many ways is moving forward with great momentum, but in other ways has not changed fundamentally. Many key advances have been made, but very often they seem to lead back to the same places

Attaching metabolic expenditures to standard occupational classification systems: perspectives from time-use research.

Traditionally, time-use data have been used to inform a broad range of economic and sociological research topics. One of the new areas in time-use research is the study of physical activity (PA) and physical activity energy expenditure (PAEE). Time-use data can be used to study PAEE by assigning MET values to daily activities using the Ainsworth Compendium of Physical Activities. Although most diarists record their daily activities accurately and in detail, they are only required to record their paid working hours, not the job-specific tasks they undertake. This makes it difficult to assign MET values to paid work episodes.In this methodological paper, we explain how we addressed this problem by using the detailed information about respondents' occupational status included in time-use survey household and individual questionnaires. We used the 2008 ISCO manual, a lexicon of the International Labour Organization of occupational titles and their related job-specific tasks. We first assigned a MET value to job-specific tasks using the Ainsworth compendium (2011) then calculated MET values for each of the 436 occupations in the ISCO-08 manual by averaging all job-specific MET values for each occupation.The ISCO-08 Major Groups of 'elementary occupations' and 'craft and related trades workers' are associated with high PAEE variation in terms of their job-specific MET values and together represented 21.6% of the Belgian working population in 2013. We recommend that these occupational categories should be prioritised for further in-depth research into occupational activity (OA).We developed a clear and replicable procedure to calculate occupational activity for all ISCO-08 occupations. All of our calculations are attached to this manuscript which other researchers may use, replicate and refine

Attaching metabolic expenditures to standard occupational classification systems: perspectives from time-use research.

Traditionally, time-use data have been used to inform a broad range of economic and sociological research topics. One of the new areas in time-use research is the study of physical activity (PA) and physical activity energy expenditure (PAEE). Time-use data can be used to study PAEE by assigning MET values to daily activities using the Ainsworth Compendium of Physical Activities. Although most diarists record their daily activities accurately and in detail, they are only required to record their paid working hours, not the job-specific tasks they undertake. This makes it difficult to assign MET values to paid work episodes.In this methodological paper, we explain how we addressed this problem by using the detailed information about respondents' occupational status included in time-use survey household and individual questionnaires. We used the 2008 ISCO manual, a lexicon of the International Labour Organization of occupational titles and their related job-specific tasks. We first assigned a MET value to job-specific tasks using the Ainsworth compendium (2011) then calculated MET values for each of the 436 occupations in the ISCO-08 manual by averaging all job-specific MET values for each occupation.The ISCO-08 Major Groups of 'elementary occupations' and 'craft and related trades workers' are associated with high PAEE variation in terms of their job-specific MET values and together represented 21.6% of the Belgian working population in 2013. We recommend that these occupational categories should be prioritised for further in-depth research into occupational activity (OA).We developed a clear and replicable procedure to calculate occupational activity for all ISCO-08 occupations. All of our calculations are attached to this manuscript which other researchers may use, replicate and refine

Additional file 1: of Attaching metabolic expenditures to standard occupational classification systems: perspectives from time-use research

This file contains – in its separate tabs – the different steps of the calculation procedure we outlined in paragraph ‘4.1 An alternative approach to applying MET values to occupational codes’. The first tab ‘ISCO - METs calculation’ provides a list of all 436 four-digit ISCO-08 codes and their underlying job-specific tasks. We assigned MET values to these underlying job-specific tasks using our ‘task abbreviations/task component calculations’ list, which we present in the second tab ‘Job-specific tasks’. The METs assigned to this list were based on the most recent version of the Ainsworth compendium [23], which we outlined in the third tab ‘Compendium - Ainsworth (2011)’. The fourth and final tab ‘ISCO - METs table’ presents an overview of all four-digit ISCO-08 codes and their assigned MET values. (XLSX 522 kb

SAXS Analysis of the tRNA-Modifying Enzyme Complex MnmE/MnmG Reveals a Novel Interaction Mode and GTP-Induced Oligomerization

Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm5U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle