Biology subjects

Kleffmann, T.

Publications and source records attributed to Kleffmann, T..

4 recordsLinked to original sources

bioRxiv2022

Quantitative comparison of manuka and clover honey proteomes with royal jelly

Royal jelly and honey are two substances produced successively by the worker bee caste. Sequential window acquisition of all theoretical fragment-ion spectra mass spectrometry (SWATH-MS) was used to compare protein quantities of bee origin in manuka and clover honey to royal jelly. Two analysis techniques identified 75 proteins in total. Peptide intensity was directly compared for a subset of 32 proteins that were identified with high confidence, and the relative changes in protein abundance were compared between each honey and royal jelly. A total of 16 proteins were found only in honey, 9 of which were common to manuka and clover honeys, and 7 found only in manuka honey. None of the identified proteins were unique to clover honey, while four proteins were unique to royal jelly. Major Royal Jelly Proteins (MRJPs) had similar profiles in both honeys, except MRJP6, which was significantly more abundant in clover honey. Quantitative data suggests changes of the protein composition as bees develop through the worker caste, with subtle changes in MRJP amounts, and extensive changes in proteins with enzymatic function. Metabolism enzymes were more abundant in honey than in royal jelly, where the trend revealed a potential catalytic role for two uncharacterised proteins (MRJP6 and LOC408608). Differences in the proteomes of the two types of honey probably reflects differences in the type of nectar foraged.

molecular biology↗

bioRxiv2022

A lipopolysaccharide-dependent phage infects a pseudomonad phytopathogen and can evolve to evade phage resistance

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of {Phi}Psa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of {Phi}Psa374, we revealed the structural proteome and that this phage possess a T=9 capsid triangulation, unusual for myoviruses. Furthermore, we show that {Phi}Psa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage-resistance is overcome by mutations in a tail fiber and structural protein of unknown function in {Phi}Psa374. This study provides new insight into the properties of {Phi}Psa374-like phages that informs their use as antimicrobials against Psa. Originality-Significance StatementThe rise of phytopathogen resistance to agrichemicals poses a significant threat to crop production, and requires urgent attention. The work presented here examines a phage genus, members of which utilize LPS as a receptor, and show potential as biocontrol agents of Psa. Studies in planta showed the development of Psa resistance to the phage, whilst maintaining bacterial virulence. This finding underscores the importance of examining phages in their ecological context for the informed design of phage cocktails that are effective in phytopathogen control.

microbiology↗

bioRxiv2021

Oncogenic truncations of ASXL1 enhance a motif for BRD4 ET-domain binding

Proper regulation of gene-expression relies on specific protein-protein interactions between a myriad of epigenetic regulators. As such, mutation of genes encoding epigenetic regulators often drive cancer and developmental disorders. Additional sex combs-like protein 1 (ASXL1) is a key example, where mutations frequently drive haematological cancers and can cause developmental disorders. It has been reported that nonsense mutations in ASXL1 promote an interaction with BRD4, another central epigenetic regulator. Here we provide a molecular mechanism for the BRD4-ASXL1 interaction, demonstrating that a motif near to common truncation breakpoints of ASXL1 contains an epitope that binds the ET domain within BRD4. Binding-studies show that this interaction is analogous to common ET-binding modes of BRD4-interactors, and that all three ASX-like protein orthologs (ASXL1-3) contain a functional ET-domain-binding epitope. Crucially, we observe that BRD4-ASXL1 binding is markedly increased in the prevalent ASXL1Y591X truncation that maintains the BRD4-binding epitope, relative to full-length ASXL1 or truncated proteins that delete the epitope. Together, these results show that ASXL1 truncation enhances BRD4 recruitment to transcriptional complexes via its ET domain, which could misdirect regulatory activity of either BRD4 or ASXL1 and may inform potential therapeutic interventions.

biochemistry↗

bioRxiv2020

Critical role for cold shock protein YB-1 in cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and cancer progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cell lines resulted in cytokinesis failure, multinucleation and an increase in G1 transit time. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging of cells undergoing cytokinesis both in vitro and in zebrafish embryos, we found that YB-1 was critical for microtubule organization during cytokinesis. Using mass spectrometry we identified multiple novel phosphorylation sites on YB-1. We show that phosphorylation of YB-1 at multiple serine residues was essential for its function during cytokinesis. Using atomistic modelling we show how multiple phosphorylations alter YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining for the first time precisely how YB-1 regulates cell division. SUMMARYY-box-binding protein-1, YB-1, is essential for cell division, but it is not clear how it functions. Using live imaging and confocal microscopy we show that YB-1 functions only in the last step of division, specifically being required to initiate cytokinesis.

cell biology↗