Biochemistry and Chemical Biology

eLife assessment

This manuscript describes useful information on the interactions of the BRAF N-terminal regulatory regions (CRD, RBD and BSR) with the C-terminal kinase domain and with the upstream regulators HRAS and KRAS. The authors provide solid evidence that the BRAF BSR domain may negatively regulate RAS binding and propose that the presence of the BSR domain in BRAF provides an additional layer of autoinhibitory constraints. The data will be of interest for researchers in the RAS/RAF and general kinase regulation fields.

eLife assessment

This study presents a valuable and elegant kinetic analysis of the GSKbeta activity as a function of phosphorylation and Axin binding - providing insights into critical steps of Wnt pathway signaling. The results will be of big use to the broader signaling community, however, the incomplete dissection of the mechanism by which Axin binding inhibits GSKbeta inhibitory phosphorylation remains a weakness of this study. The work will be of broad interest to cell biologists and biochemists.

eLife assessment

The manuscript represents an important contribution to an ongoing discussion about the substrate binding site and mechanism of the Bile Acid Sodium Symporter (BASS) family of transporters. Structural and biochemical analysis of a bacterial homolog, ASTBnm, in complex with its native substrate (not bile acids, but a vitamin A precursor, pantoate) show a new binding site that is consistent with classical proposals for elevator-type transport mechanisms. Molecular dynamics (MD) simulations highlight the improved stability for the substrate in the active site when ions are present, suggesting a binding order during the transport cycle. The structural studies, binding assays, and MD simulations are convincing.

eLife assessment

The study provides useful data supporting prior findings that mitochondria in cultured cells maintain a temperature that is up to 15°C above the external temperature at which cultured cells are maintained. The evidence supporting the hypothesis is solid, although direct measures of temperature in isolated mitochondria or comparison with other cellular compartments would have strengthened the ability to interpret the relevance of the findings. Nevertheless, the bioenergetic implications of the work will be of interest to cell biologists, biochemists, and physiologists.

eLife assessment

This study is useful in identifying several gain-of-function division variants that can suppress the elongated cell division defect phenotype caused by the depletion of Age1 (or AdvA as named in other studies), a known essential cell division protein in Acinetobacter baumannii. However, characterizations of AdvA's localization patterns and its interactions with other divisome proteins are incomplete due to the lack of (1) functional characterizations of fluorescent fusion proteins, (2) considerations for membrane protein topology in the bacterial two hybrid assay, and (3) lack of high-quality fluorescence images for the co-localization studies. The results do not yet support the major claim that Age1 plays a critical role in the assembly of the A. baumannii divisome.

eLife assessment

This study presents a novel fluorescence based imaging strategy to investigate the folding status of RNA-binding proteins (RBPs) and their association with molecular chaperones under stress. It provides fundamental findings that will potentially advance our understanding in the folding and aggregation status of RBPs in nuclear stress bodies in a significant manner. However, there is also the concern that the evidence regarding protein fate is incomplete and additional controls are needed to fully support the conclusion. The imaging methodology can be adapted to study many other proteins that undergo liquid-liquid phase separation under specific cellular conditions.

eLife assessment

The manuscript describes the synergy among PI3Kbeta activators, providing compelling results concerning the mechanism of their activation. The particular strengths of the work arise to a great extend from the reconstitution system better mimicking the natural environment of the plasma membrane than previous setups have. The study will be a landmark contribution to the signaling field.

eLife assessment

This manuscript describes a fundamental strategy for developing isozyme-selective inhibitors of inositol hexakisphosphate kinases. The compelling evidence that subtle changes to the gatekeeper position can sensitize the inositol hexakisphosphate kinase mutant to allosteric inhibitors will undoubtedly inspire other analog-sensitive inhibitor studies. This manuscript will be of interest to researchers focusing on kinase regulation and inhibitor design.

eLife assessment

This is important work that examines hepatic acetate production via ACOT12/18 in starvation and diabetes. The investigators use solid loss of function strategies in cells, including mouse primary hepatocytes, and in vivo mouse experiments to show that ACOTs are necessary for normal acetate production in the context of fasting and type 1 diabetes. Given that acetate is commonly thought to primarily represent a fermentation product, this study is of interest as it describes hepatic pathways converting fatty acids to acetate.

eLife assessment

This study presents valuable structures of a pentameric ligand-gated ion channel from a thermophilic worm that is a homologue of the well-known mammalian nicotinic receptors. Although the function of the worm receptor is unknown, the authors convincingly identify interesting features for this class of receptors including a steroid detergent that is bound in the canonical neurotransmitter site and that induces conformational changes of the extracellular domains. These observations will be of broad interest to the ligand-gated ion channel community, although it is difficult at this moment to relate these observations to channel function as the channel's activating ligand remains unknown.

eLife assessment

Dhekne et al report a novel pathway for activation of the multi-domain LRRK2 protein kinase by Rab12 GTPase. LRRK2, which is mutated in Parkinson's Disease phosphorylates a subset of Rab proteins involved in intracellular trafficking, and Parkinson's disease-linked mutations increase this phosphorylation. This work adds an important new layer of understanding of this highly complex pathway by revealing that LRRK2's binding to Rab12 enhances its ability to phosphorylate Rab10. This conclusion is supported by compelling evidence from a wide array of rigorous approaches.

eLife assessment

This important study makes use of AlphaFold2 to predict the models of tens of cohesin subcomplexes from different species. The models, which are in most cases consistent with published cohesin variants with compromised in vitro and in vivo cohesin activity, provide convincing evidence that leads to testable hypotheses of cohesin dynamics and regulation. More broadly, this study serves as an example of how to use AlphaFold2 to build models of protein complexes that involve the docking of flexible regions to globular domains.

eLife assessment

The authors provide a fundamental resource, detailing genetic variation of nutrient-responsive islet calcium regulation in mice through the lens of proteomics. The evidence for the mechanisms identified using this resource is compelling and strongly supported by integration with results from genome-wide association studies in humans. The construction of a streamlined and searchable web interface for the data will maximize their accessibility and utilization by the community.

eLife assessment

In this work, the authors provide important mechanistic insights into how the intracellular effector protein Calcineurin B homologous protein 3 (CHP3) can be regulated in a calcium-independent manner to expose its lipid binding site. Compelling evidence demonstrates a binding partner protein (NHE1) triggers a conformation change and exposure of the myristoyl group in CHP3 resulting in membrane association. This provides mechanistic insight into the signalling mechanisms achieved by CHP3 in a target-dependent manner, which will be of broad scientific interest.

eLife assessment

The manuscript reports useful findings by resolving the crystal structure of Sedoheptulose-1,7-Bisphosphatase (SBPase) from the green algae Chlamydomonas reinhardtii, which is involved in the Calvin cycle. The data presented are solid based on validated methodologies, which help in understanding the structure and function of this enzyme.

eLife assessment

This study presents valuable findings on the organization of respiratory chain complexes in mitochondria. It provides solid evidence that respiratory supercomplex formation in the fruit fly does not impact respiratory function, suggesting the role of these complexes is structural, rather than catalytic. However, whether the conclusions extend to other species requires further evidence. This manuscript will be of broad interest to the field of mitochondrial bioenergetics.

eLife assessment

This study reports a new approach to determine the architecture of peptidoglycan (PG), the primary component of the bacterial cell wall, validating the pipeline through an architectural analysis of several members of the human gut microbiota. The technique is potentially valuable for this sub-field as it would enable researchers interested in peptidoglycan in a range of organisms to easily assess muropeptide composition in an easy, automated manner. However, there is some uncertainty about whether the pipeline was fully automated and it was noted that the pipeline requires prior knowledge of the peptidoglycan composition of an organism. Additionally, the use of the technique to investigate whether PG cross-bridge length is a determinant of cell wall stiffness produced evidence that would need more direct support and is therefore so far incomplete.

eLife assessment

This manuscript reports important findings, demonstrating a critical role for a cysteine-containing dimerization interface in the secretion of FGF2 through an unconventional pathway. The authors provide compelling evidence, combining in vitro biochemical assays with structural simulation. The work will be of interest to researchers working on protein trafficking and secretion.

eLife assessment

This study provides useful insights into the mechanisms of electron transport in STEAP proteins, consistent with current models. The work strengthens and supports previously published biochemical and structural data, and the experimental results are of solid technical quality. The manuscript will be of interest to colleagues who work on STEAP proteins and related electron transfer systems.

eLife assessment

This study presents fundamental new insight into the regulatory apparatus of PI3Kgamma; an important kinase in signaling pathways that control the immune response and cancer. A suite of biophysical and biochemical approaches provide convincing evidence for new sites of allosteric control over enzyme activity. The rigorous findings provide structure and dynamic information that may be exploited in efforts to control PI3Kgamma activity in a therapeutic setting.