Biochemistry

Systematic characterization of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry

1. Hao Jiang
2. Jimena Druker
3. James W Wilson
4. Dalila Bensaddek
5. Jason R Swedlow
6. Sonia Rocha
7. Angus I Lamond

• Curated by eLife

  eLife Assessment

  The study presents a valuable resource of proline hydroxylation proteins for molecular biology studies in oxygen-sensing and cell signaling with the characterization of Repo-man proline hydroxylation site. The evidence supporting the claim of the authors is solid, although further clarification of the overall efficiency of the HILIC analysis, the specificity/sensitivity of immonium ion analysis, as well as quantification of proline hydroxylation identifications will be helpful. The work will be of interest to researchers studying post-translational modification, oxygen sensing, and cell signaling.

Reviewed by eLife

Determining the off-target activity of antibiotics and novel translation initiation sites in mitochondria

1. James Marks
2. Emma Young
3. Markus Hafner

• Curated by eLife

  eLife Assessment

  This valuable study presents findings on how prokaryotic antibiotics affect translation in mitochondrial ribosomes. Using mitoribosome profiling, the authors provide solid evidence that most tested antibiotics act similarly on bacterial and mitochondrial translation. Additionally, this work shows that alternative translation initiation events might exist in two specific mt-mRNAs (MT-ND1 and MT-ND5). However, additional biochemical and structural experiments are needed to support these findings.

Reviewed by eLife

Single-step in vitro ribosome reconstitution mediated by two GTPase factors, EngA and ObgE

1. Aya Sato
2. Weng Yu Lai
3. Yusuke Sakai
4. Yoshihiro Shimizu

• Curated by eLife

  eLife Assessment

  This is a useful study presenting solid data indicating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions. The study elegantly bridges the gap between the non-physiological aspects of the previous two-step reconstitution method and the extract-dependent iSAT system to enable ribosome assembly under translation-compatible conditions; however, it is limited by reliance on rRNA and proteins extracted from native ribosomes and does not achieve a true bottom-up reconstruction from all synthetic components. The evidence is incomplete in not characterizing the spectrum of reporter polypeptides produced and not comparing their rate and yield of synthesis from reconstituted ribosomes to that obtained with pure native ribosomes; and the impact of the study is limited by not including reporters to examine the fidelity of initiation, elongation or termination achieved with the reconstituted ribosomes.

Reviewed by eLife

Automated genome mining predicts structural diversity and taxonomic distribution of peptide metallophores across bacteria

1. Zachary L Reitz
2. Bita Pourmohsenin
3. Melanie Susman
4. Emil Thomsen
5. Daniel Roth
6. Alison Butler
7. Nadine Ziemert
8. Marnix H Medema

• Curated by eLife

  eLife Assessment

  This important and compelling study establishes a robust computational and experimental framework for the large-scale identification of metallophore biosynthetic clusters. The work advances beyond current standards, providing theoretical and practical value across microbiology, bioinformatics, and evolutionary biology.

Reviewed by eLife

Purified Zymogens Reveal Mechanisms of Snake Venom Metalloproteinase Auto-Activation

1. Sophie Hall
2. Iara Aimê Cardoso
3. Mark C Wilkinson
4. Maria Molina Carretero
5. Srikanth Lingappa
6. Bronwyn Rand
7. Dakang Shen
8. Johara Boldrini-França
9. Richard Stenner
10. Stefanie K Menzies
11. Georgia Balchin
12. Konrad Kamil Hus
13. Renaud Vincentelli
14. Andrew Mumford
15. Nicholas R Casewell
16. Imre Berger
17. Christiane Schaffitzel

• Curated by eLife

  eLife Assessment

  In this useful paper, the authors present a comprehensive method for the purification of recombinant Snake Venom Metalloproteinases (SVMPs) using the MultiBac expression system, explain the self-activation of the enzymes by Zn2+ incubation, and establish high-throughput screening (HTS) techniques. The authors addressed a key problem: producing a substantial amount of pure and enzymatically active SVMPs required for structural and functional studies. Altogether, this work builds a solid foundation for the large-scale production of active SVMPs for future biochemical and structural characterization as well as for drug discovery, albeit leaving certain caveats about the universal applicability of the described methodology for the production of any recombinant SVMPs.

Reviewed by eLife

Structure of the human CTF18–RFC clamp loader bound to PCNA

1. Giuseppina R Briola
2. Muhammad Tehseen
3. Amani Al-Amodi
4. Grace Young
5. Ammar U Danazumi
6. Phong Quoc Nguyen
7. Christos G Savva
8. Mark Hedglin
9. Samir M Hamdan
10. Alfredo De Biasio

• Curated by eLife

  eLife Assessment

  This paper reports new data on the structure of the human CTF18-RFC clamp loader complex bound to the PCNA clamp. The new and convincing data compliment previous reports of CTF-RFC-PCNA structures and as such, represents an important contribution.

Reviewed by eLife

Common and distinct roles of AMPKγ isoforms in small-molecule activator-stimulated glucose uptake in mouse skeletal muscle

1. Dipsikha Biswas
2. Ever Espino-Gonzalez
3. Danial Ahwazi
4. Jordana B. Freemantle
5. Charline Jomard
6. Jonas Brorson
7. Agnete N. Schou
8. Jean Farup
9. Julien Gondin
10. Jesper Just
11. Marc Foretz
12. Jonas T. Treebak
13. Marianne Agerholm
14. Kei Sakamoto

Reviewed by PREreview

Cryo-EM reveals multiple mechanisms of ribosome inhibition by doxycycline

1. William S. Stuart
2. Michail N. Isupov
3. Mathew McLaren
4. Christopher H. Jenkins
5. Adam Monier
6. Bertram Daum
7. Isobel H. Norville
8. Vicki A.M. Gold
9. Nicholas J. Harmer

Reviewed by preLights

A sulfatide-centered ultra-high resolution magnetic resonance MALDI imaging benchmark dataset for MS1-based lipid annotation tools

1. Lars Gruber
2. Stefan Schmidt
3. Thomas Enzlein
4. Carsten Hopf

Reviewed by GigaScience

Shifting the PPARγ conformational ensemble toward a transcriptionally repressive state improves covalent inhibitor efficacy

1. Liudmyla Arifova
2. Brian S MacTavish
3. Zane Laughlin
4. Mithun Nag Karadi Girdhar
5. Jinsai Shang
6. Min-Hsuan Li
7. Xiaoyu Yu
8. Di Zhu
9. Theodore M Kamenecka
10. Douglas J Kojetin

• Curated by eLife

  eLife Assessment

  This manuscript presents a fundamental advance in our understanding of nuclear receptor pharmacology by expanding on previous work demonstrating dual ligand occupancy in the peroxisome proliferator-activated receptor-gamma (PPARγ). Using a compelling combination of biophysical, biochemical, and cellular approaches, the authors show that covalent inverse agonists with enhanced efficacy shift the receptor conformation toward a transcriptionally repressive state that limits orthosteric ligand co-binding more effectively. This revised manuscript further strengthens support for a proximal, bidirectional allosteric model of dual ligand occupancy by sharpening the distinction between prior and new findings, adding clear conceptual figures, and strengthening statistical rigor.

Reviewed by eLife