Biochemistry

Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism

1. Dylan Gerard Ryan
2. Ming Yang
3. Hiran A Prag
4. Giovanny Rodriguez Blanco
5. Efterpi Nikitopoulou
6. Marc Segarra-Mondejar
7. Christopher A Powell
8. Tim Young
9. Nils Burger
10. Jan Lj Miljkovic
11. Michal Minczuk
12. Michael P Murphy
13. Alex von Kriegsheim
14. Christian Frezza

• Curated by eLife

  Evaluation Summary:

  The authors of this study investigate the consequences of acute or chronic disruption of parts of the TCA cycle, and how different interventions can drive different transcriptional responses. Specifically, the authors use both pharmacological and genetic methods to disrupt succinate dehydrogenase or fumarate hydratase, and characterize the effect of each on metabolism. They also find that disruption of these enzymes elicits a transcriptional response through ATF4. This work provides insight into how metabolism is affected by TCA cycle loss, and how how this affects metabolic stress signaling.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

Reviewed by eLife

Disintegration promotes protospacer integration by the Cas1-Cas2 complex

1. Chien-Hui Ma
2. Kamyab Javanmardi
3. Ilya J Finkelstein
4. Makkuni Jayaram

• Curated by eLife

  Evaluation Summary:

  In this manuscript, an in vitro Cas1-Cas2 model system is used to study the reaction used to insert foreign DNA elements into a CRISPR array during the adaptive phase of immunity. The authors propose that hydrolysis of one end of the transposon DNA may be the primary mechanism for the insertion of very small DNA elements (which are difficult to bend tightly) that are found for the proto spacer sequences, and that cellular repair pathways are responsible for ligating the CRISPR array back together in vivo. The findings additionally suggest that water-mediated disintegration has an unappreciated role in the generation of CRISPR arrays as part of the bacterial immune response. These hypotheses are intriguing and of potential interest to those in the CRISPR field. However, it is unclear how this in vitro study, which does not monitor the full the reaction (directionality is lost due to the lack of a PAM sequence in the substrate and several required cellular factors are missing), relates to transposition as it occurs in vivo. Overall, this is an interesting study that challenges the current thinking in the field, but it does not present sufficient evidence to establish the physiological significance of the observed effects, thereby limiting its potential broader impact.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by eLife

Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation

1. Jesse M Hansen
2. Avital Horowitz
3. Eric M Lynch
4. Daniel P Farrell
5. Joel Quispe
6. Frank DiMaio
7. Justin M Kollman

• Curated by eLife

  Evaluation Summary:

  This work provides valuable new information to those who study enzyme mechanisms, nucleotide metabolism, and the response of cells to stress such as nutrient deprivation. The study focuses on CTP Synthase (CTPS), an important enzyme in nucleotide biosynthesis that has been shown to assemble into foci and filaments in yeast cells undergoing starvation conditions. The authors study the structure of yeast CTPS and its propensity to polymerize in low pH (mimicking starvation conditions), and how CTPS filamentation relates to the cellular assemblies.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Allosteric cooperation in β-lactam binding to a non-classical transpeptidase

1. Nazia Ahmad
2. Sanmati Dugad
3. Varsha Chauhan
4. Shubbir Ahmed
5. Kunal Sharma
6. Sangita Kachhap
7. Rana Zaidi
8. William R Bishai
9. Gyanu Lamichhane
10. Pankaj Kumar

• Curated by eLife

  Evaluation Summary:

  This manuscript reports high-resolution crystallographic structures of the L,D, transpeptidase from Mycobacterium tuberculosis. These structures were obtained with ligands (a sugar molecule and a beta-lactam). A surprising finding is that the enzyme contains a ligand-binding site located greater than 20 Å away from the catalytic site. The authors propose and provide some evidence for an allosteric role of the new ligand site (S-pocket), which would be significant because it could allow new ways of targeting the protein for inhibition. While enthusiasm is high for the discovery of a putative allosteric site, more rigorous computation is necessary, along with some biochemical investigations and mutagenesis studies to rule out the possibility of a different role for the S-site. Moreover, a better articulation of the connection/crosstalk between the two sites in the form of a mechanistic hypothesis would strengthen the paper.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by eLife

Assessing target engagement using proteome-wide solvent shift assays

1. Jonathan G Van Vranken
2. Jiaming Li
3. Dylan C Mitchell
4. José Navarrete-Perea
5. Steven P Gygi

• Curated by eLife

  Evaluation Summary:

  This manuscript will be of broad interest to readers in the field of proteomics and drug discovery. It describes a potentially robust method for the identification of biological targets of small molecules, a substantial hurdle in drug discovery. The experiments described are rigorous and this manuscript provides a useful template for the broad implementation of this method. One conclusion that needs further support is the one of the complementarity of CPP and TPP (as in "these two approaches share much in common, they remain distinct and likely serve to complement one another").

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by eLife

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir

1. Calvin J. Gordon
2. Hery W. Lee
3. Egor P. Tchesnokov
4. Jason K. Perry
5. Joy Y. Feng
6. John P. Bilello
7. Danielle P. Porter
8. Matthias Götte

Reviewed by ScreenIT

Rapid and Effective Inactivation of SARS-CoV-2 with a Cationic Conjugated Oligomer with Visible Light: Studies of Antiviral Activity in Solutions and on Supports

1. Kemal Kaya
2. Mohammed Khalil
3. Benjamin Fetrow
4. Hugh Fritz
5. Pradeepkumar Jagadesan
6. Virginie Bondu
7. Linnea Ista
8. Eva Y. Chi
9. Kirk S. Schanze
10. David G. Whitten
11. Alison Kell

Reviewed by ScreenIT

Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single DNA Lesions

1. Ana H. Sales
2. Iwen Fu
3. Alexander Durandin
4. Sam Ciervo
5. Tania J. Lupoli
6. Vladimir Shafirovich
7. Suse Broyde
8. Nicholas E. Geacintov

Reviewed by ScreenIT

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

1. Aixin Song
2. Zachary Hazlett
3. Dulith Abeykoon
4. Jeremy Dortch
5. Andrew Dillon
6. Justin Curtiss
7. Sarah Bollinger Martinez
8. Christopher P Hill
9. Clinton Yu
10. Lan Huang
11. David Fushman
12. Robert E Cohen
13. Tingting Yao

• Curated by eLife

  Evaluation Summary:

  This manuscript addresses the role of the deubiquitylating enzyme UCH37 in facilitating proteasomal clearance of branched polyubiquitylated substrates. Using a wide-range of chemical biological, biophysical and cell biological techniques, the authors have convincingly demonstrated that UCH37 binds to branched ubiquitin trimers, with at least one K48 linkage, by binding to both distal ubiquitins attached to the proximal, or central, ubiquitin. They further demonstrate that mutations of UCH37 lead to the formation of proteasomal foci in cells and that these foci are rich in polyubiquitinated species, presumably due to the lack of debranching by UCH37. Overall, this excellent study adds to our understanding of UCH37 function, especially with regard to the newly observed phenomenon of reversible proteasome aggregation in cells. Readers will benefit from the large array of ubiquitin-centric tools that are described to study key aspects of UCH37 function and from knowledge of the specific role of UCH37.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by eLife

Structure and ion-release mechanism of PIB-4-type ATPases

1. Christina Grønberg
2. Qiaoxia Hu
3. Dhani Ram Mahato
4. Elena Longhin
5. Nina Salustros
6. Annette Duelli
7. Pin Lyu
8. Viktoria Bågenholm
9. Jonas Eriksson
10. Komal Umashankar Rao
11. Domhnall Iain Henderson
12. Gabriele Meloni
13. Magnus Andersson
14. Tristan Croll
15. Gabriela Godaly
16. Kaituo Wang
17. Pontus Gourdon

• Curated by eLife

  Evaluation Summary:

  This paper presents crystal structures of sCoaT, a heavy metal transporting P-type ATPase. These structures and complementary functional data define the overall fold of this protein and provide insight into several mechanistic features, including a conserved histidine proposed to act as a novel counter-ion during transport. The study will be of interest to biochemists and microbiologists interested in the transport of transition metals, structural biology of membrane proteins and drug development.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by eLife