Biochemistry

A Membrane-Disruptive Action of VBIT-4 Challenges Its Role as a Widely Used VDAC1 Oligomerization Inhibitor

1. Varun Ravishankar
2. Luís Borges-Araújo
3. Megha Rajendran
4. Elodie Lafargue
5. Deborah Byrne
6. Nicolas Buzhinsky
7. Mya S Wolfe
8. Wendy Fitzgerald
9. Nina A Bautista
10. Bethel G Beyene
11. Motahareh G Larimi
12. Jean-Pierre Duneau
13. James Sturgis
14. Sergey M Bezrukov
15. Ignacio Casuso
16. Tatiana Rostovtseva
17. Lucie Bergdoll

• Curated by eLife

  eLife Assessment

  This work provides a reassessment of VBIT-4, a compound previously proposed to inhibit oligomerization of the crucial protein known as the mitochondrial voltage-dependent anion channel. Combining complementary experimental approaches with molecular dynamics simulations, the authors provide compelling evidence that VBIT-4 primarily disrupts lipid membranes and induces channel-independent cytotoxicity. The study has important implications for interpreting previous work using VBIT-4 as a probe of channel function and highlights the need to consider membrane-disruptive effects when evaluating drug mechanisms.

Reviewed by eLife

A simplified and highly efficient cell-free protein synthesis system for prokaryotes

1. Xianshengjie Lang
2. Changbin Zhang
3. Jingxuan Lin
4. Zhe Zhang
5. Wenfei Li

• Curated by eLife

  eLife Assessment

  The study presents valuable findings of a new E. coli cell-free protein synthesis (eCFPS) system that has been simplified by reducing the number of core components from 35 to 7; furthermore, the findings communicate a simplified 'fast lysate' preparation that eliminates the need for traditional runoff and dialysis steps. It is interesting that the system's robustness is exhibited by its applicability to nanoluc, a protein that expresses readily in many systems, to more challenging proteins like the functional self-assembling vimentin and the active restriction endonuclease Bsal. Despite the study representing an advancement towards simplifying protein expression workflows, the evidence is solid and supports the main claims however minor weakness exists i.e. the efficiency claims about the new system needs to be supported by accurate comparisons with typical cell free expression systems, in addition, investigations into the mechanistic basis of the observations would provide more evidence. Despite this shortcoming, the paper remains of interest to scientists in cell and molecular biology, microbiology, biotechnology and protein synthesis.

Reviewed by eLife

Profiling of terminating ribosomes reveals translational control at stop codons

1. Longfei Jia
2. Yuanhui Mao
3. Saori Uematsu
4. Xinyi Ashley Liu
5. Leiming Dong
6. Leonardo Henrique França de Lima
7. Shu-Bing Qian

• Curated by eLife

  eLife Assessment

  This manuscript reports on the application of ribosome profiling (EZRA-seq and eRF1-seq) and massively parallel reporter assays (MPRA) to identify and characterize sequence elements that regulate translation termination. The authors conclude that a GA-rich element upstream of stop codons is associated with ribosome pausing during translation termination; in contrast, C-rich sequences upstream of stop codons abolish termination pausing. While the overall findings of this study are useful and the identification of GA-rich elements upstream of stop codons is compelling, support for several other claims remains incomplete. Specifically, the evidence that the MPRA results mirror the ribosome profiling, that a C-rich sequence preceding the stop codon promotes termination slippage in cellular mRNAs, and that Rps26 interferes with mRNA interactions to regulate translation termination would benefit from further support.

Reviewed by eLife

The chromokinesin Kid (KIF22) forms a homodimer, moves processively along microtubules and transports double-stranded DNA

1. Shinsuke Niwa
2. Natsuki Furusaki
3. Tomoki Kita
4. Yuki Suzuki
5. Kyoko Chiba

• Curated by eLife

  eLife Assessment

  This important study clarifies the mechanism by which the kinesin-10 motor protein, chromosome-associated kinesin, Kid (KIF22), enables chromosome movement during mitosis, demonstrating that human and Xenopus Kid proteins function as processive, homodimeric kinesins capable of processive microtubule plus-end motility. The convincing work highlights that Kid can recruit and transport duplex DNA along microtubules via its conserved C-terminal DNA binding domain, revising our understanding of chromokinesins' role in chromosome motility during mitosis. It will be of interest to those in the molecule motor community working at the molecular, cellular, and organismal levels.

Reviewed by eLife

Cardiolipin deficiency disrupts electron transport chain and drives steatohepatitis

1. Marisa J Brothwell
2. Guoshen Cao
3. J Alan Maschek
4. Annelise M Poss
5. Alek D Peterlin
6. Liping Wang
7. Talia B Baker
8. Justin L Shahtout
9. Piyarat Siripoksup
10. Quentinn J Pearce
11. Jordan M Johnson
12. Fabian M Finger
13. Alexandre Prola
14. Sarah A Pellizzari
15. Gillian L Hale
16. Allison M Manuel
17. Shinya Watanabe
18. Edwin R Miranda
19. Kajsa E Affolter
20. Trevor S Tippetts
21. Linda S Nikolova
22. Ran Hee Choi
23. Stephen T Decker
24. Mallikarjun Patil
25. J Leon Catrow
26. William L Holland
27. Sara M Nowinski
28. Daniel S Lark
29. Kelsey H Fisher-Wellman
30. Patrice N Mimche
31. Kimberley J Evason
32. James E Cox
33. Scott A Summers
34. Zach Gerhart-Hines
35. Katsuhiko Funai

• Curated by eLife

  eLife Assessment

  This revised manuscript retreats from the original claim of establishing a causal link between cardiolipin deficiency and the progression from steatotic liver disease to steatohepatitis and instead advances a more limited mechanistic conclusion: that cardiolipin deficiency perturbs electron transport and promotes electron leak from the mitochondrial respiratory chain. The experimental evidence supporting this revised claim is now solid, and the potential for increased electron leak to contribute to liver pathophysiology is demonstrated. However, absent evidence that cardiolipin deficiency is causally upstream of disease progression, the overall significance of the work remains limited. While the study provides a convincing analysis of mitochondrial bioenergetics, the narrowing of its central claim diminishes its impact relative to that proposed in the original submission.

Reviewed by eLife

Dynamic assembly of malate dehydrogenase–citrate synthase multienzyme complex in the mitochondria

1. Joy Omini
2. Inga Krassovskaya
3. Taiwo Adeolu Dele-Osibanjo
4. Connor Pedersen
5. Toshihiro Obata

• Curated by eLife

  eLife Assessment

  This important study provides novel information on multi-enzyme complexes, known as metabolons, that form between sequential enzymes in a metabolic pathway. Using an innovative NanoBiT split-luciferase system, the authors present compelling evidence that malate dehydrogenase (MDH1) and citrate synthase (CIT1) dynamically associate under different metabolic conditions in Saccharomyces cerevisiae. The findings suggest the dynamic MDH1-CIT1 interaction facilitates control of TCA pathway flux rate.

Reviewed by eLife

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

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

• Curated by eLife

  eLife Assessment

  This is a valuable study presenting convincing 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 assembly of highly functional ribosomes under translation-compatible conditions. The reported findings represent substantial progress towards achieving a bottom-up reconstruction of the translation machinery from synthetic parts.

Reviewed by eLife

Integrating computational protein structure predictions and genetic dependencies yields an atlas of human multi-protein complexes (AHMPC)

1. Michael Uckelmann
2. Daniel Alvarez Salmoral
3. Carmen Maiella
4. Onno Bleijerveld
5. Ceri Zwart
6. Eric Marcus
7. Joren Brunekreef
8. Jonas Teuwen
9. Roderick Beijersbergen
10. Anastassis Perrakis

Reviewed by Review Commons

Intraflagellar transport protein IFT172 contains a C-terminal ubiquitin-binding U-box-like domain involved in ciliary signaling

1. Nevin K Zacharia
2. Stefanie Kuhns
3. Niels Boegholm
4. Anni Christensen
5. Jiaolong Wang
6. Narcis A Petriman
7. Anna Lorentzen
8. Jindriska L Fialova
9. Lucie Menguy
10. Sophie Saunier
11. Søren Tvorup Christensen
12. Jens S Andersen
13. Sagar Bhogaraju
14. Esben Lorentzen

• Curated by eLife

  eLife Assessment

  This important work advances our understanding of intraflagellar transport, ciliogenesis, and ciliary-based signaling, by identifying the interactions of IFT172 with IFT-A components, ubiquitin-binding, and ubiquitination, mediated by IFT172 C-terminus and its role in ciliogenesis and ciliary signaling. The evidence supporting the findings is convincing. This paper will be of interest to cell biologists and biochemists, especially those working on cilia and signaling.

Reviewed by eLife

Purified zymogens reveal mechanisms of snake venom metalloproteinase auto-activation

1. Sophie Hall
2. Iara Aime 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 Kate Menzies
11. Georgia Balchin
12. Konrad Kamil Hus
13. Renaud Vincentelli
14. Andrew Mumford
15. Alastair Poole
16. Nicholas R Casewell
17. Imre Berger
18. 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