Biology subjects

Kaschani, F.

Publications and source records attributed to Kaschani, F..

30 records · Page 2Linked to original sources

bioRxiv2023

Protein migration and topography mapping reveals endogenous protein processing and homomers in Nicotiana benthamiana.

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant protein in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as a favorite transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5,040 plant proteins of agroinfiltrated N. benthamiana within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. For instance, this analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed various processing events in vivo. This extensive proteomic dataset can be investigated further and demonstrates that most plant proteins are proteolytically processed and implicates an extensive proteolytic machinery shaping the proteome of agroinfiltrated N. benthamiana.

plant biology↗

bioRxiv2023

Ipl1-controlled attachment maturation regulates Mps1 association with its kinetochore receptor

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behaviour in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH-domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signalling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to allow anaphase onset.

cell biology↗

bioRxiv2023

The anti-tubercular callyaerins target the Mycobacterium tuberculosis-specific non-essential membrane protein Rv2113

Spread of antimicrobial resistances in the pathogen Mycobacterium tuberculosis remains a public health challenge. Thus, there is a continuous need for new therapeutic options with modes-of-action differing from current antibiotics. Previously, bioactivity-guided isolation identified the callyaerins, a class of hydrophobic cyclopeptides with an unusual (Z)-2,3-di-aminoacrylamide unit, as promising antitubercular agents. In this study, we investigated the molecular mechanisms underlying their antimycobacterial properties. Structure-activity relationship studies enabled the identification of the structural determinants relevant for their antibacterial activity. The antitubercular callyaerins are bacteriostatics selectively active against M. tuberculosis, including extensively drug-resistant (XDR) strains, with minimal cytotoxicity against human cells and a promising intracellular activity in a macrophage infection model. Via spontaneous resistance mutant screens and various chemical proteomics approaches, we showed that they act by direct targeting of the non-essential, M. tuberculosis-specific putative membrane protein Rv2113, thereby triggering a complex stress response in M. tuberculosis characterized by global downregulation of lipid biosynthesis, cell division, DNA repair and replication. Our study thus not only identifies Rv2113 as a new M. tuberculosis-specific target for antitubercular drugs, which should result in less harm of the microbiome and weaker resistance development in off-target pathogens. It furthermore demonstrates that also non-essential proteins may represent efficacious targets for antimycobacterial drugs.

microbiology↗

bioRxiv2023

TopBP1 utilises a bipartite GINS binding mode to activate the replicative helicase

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Pol{varepsilon}) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Pol{varepsilon} subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

molecular biology↗

bioRxiv2023

Glutathione transferase photoaffinity labeling demonstrates GST activation by safeners and NPR1-independent activation by BTH

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. Here, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study global GST activation in various plant species. The small molecule probe contains glutathione, a photoreactive group, and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts. Enrichment and MS analysis of labeled proteins from Arabidopsis identified ten GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST activation in wheat seedlings upon treatment with safeners, and in Arabidopsis leaves upon infection with avirulent bacteria. Photoaffinity labeling and proteomics identified six Phi- and Tau-class GSTs that are induced upon treatment with salicylic acid (SA) analog benzothiadiazole (BTH) and these were tested for enhancing immunity in disease assays. Our data confirm that BTH-induced GST activation is independent of NPR1, the master regulator of SA signaling.

plant biology↗

bioRxiv2022

Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface

The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonizing microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in leaves upon infection with Pseudomonas syringae. Using activity-based proteomics with a cocktail of biotinylated probes we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). The activity of 82 of these hydrolases (mostly SHs) increases during infection, whilst the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active {beta}-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity, and contains a E112Q active site substitution that is essential for antibacterial activity and is conserved only in Nicotiana species. This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3.

plant biology↗

bioRxiv2022

Factors governing attachment of Rhizobium leguminosarum to legume roots

Primary attachment of rhizobia to host legume roots depends on pH and is the first physical interaction during nodulation. Genome-wide insertion sequencing, luminescence-based attachment assays and proteomic analysis demonstrate primary attachment of Rhizobium leguminosarum biovar viciae 3841 to Pisum sativum (pea) roots is more complex than previously thought. In total, 115 proteins are needed for initial attachment under one or more test conditions (acid, neutral or alkaline pH), with 22 required under all conditions. These include cell-surface filamentous hemagglutinin adhesin (RL4382) and its transporter (RL4381), transmembrane protein RL2400, RL3752 (PssA, glycosyl transferase) affecting capsular polysaccharide and transcriptional regulator RL4145 (PckR). RNASeq was used to determine targets of RL4145 (PckR) and regulator RL3453. The 54 proteins required for attachment at pH 7.0 were investigated for nodulation phenotypes. Glucomannan biosynthesis protein A (GmsA) is needed at pH 6.5 and pH 7.0. Membrane proteins DgkA and ImpA are required specifically at pH 6.5, and RpoZ at pH 7.5. Sonicated cell surface fractions inhibited root attachment at alkaline pH but no overlap between proteins identified by proteomic and INseq analysis, suggests there is no single rhicadhesin needed for alkaline attachment. Our results demonstrate the complexity of primary root attachment and diversity of mechanisms involved.

microbiology↗

bioRxiv2022

Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities

Microbial communities are significant drivers of global biogeochemical cycles, yet accurate function prediction of their proteome and discerning their activity in situ for bioprospecting remains challenging. Here, we present environmental activity-based protein profiling (eABPP) as a novel proteomics-based approach bridging the gap between environmental genomics, correct function annotation and in situ enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases by combining genome-resolved metagenomics and mass spectrometry-based eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. eABPP does not only advance current methodological approaches by providing evidence for enzyme and microbial activity in situ but also represents an alternative approach to sequence homology-guided biocatalyst discovery from environmental ecosystems.

biochemistry↗

bioRxiv2022

High resolution analysis of proteolytic substrate processing

Proteolysis is a key catalytic event in protein and thus cellular homeostasis. Despite the importance and wide implications of proteolytic processing and degradation, methods describing the degradation of folded proteins at high temporal and spatial resolution are not well established. However, this information is required to obtain a deep mechanistic understanding of proteolytic events and their consequences. Here, we describe an integrated method comprising time-resolved mass spectrometry, circular dichroism spectroscopy and bioinformatics to reveal the sequential degradation and unfolding of the model substrate annexin A1 by the human serine protease HTRA1. This workflow represents a general strategy for obtaining precise molecular insights into protease-substrate interactions that can be conveniently adapted to studying other posttranslational modifications such as phosphorylation in dynamic protein complexes.

biochemistry↗

bioRxiv2020

Proteome-wide Profiling of RNA-Binding Protein Responses to flg22 Reveals Novel Components of Plant Immunity

RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation and are known to contribute to plant immunity. To understand the responses of cellular RBPs to an immune elicitor, we applied RNA interactome capture to Arabidopsis leaves treated with flg22. Strikingly, flg22 induced a pervasive remodelling of the cellular RBPome affecting 186 proteins. Flg22-responsive RBPs included classical RBPs involved in RNA metabolism as well as non-canonical RBPs. RBP responders detected after 2h of treatment are enriched in putative sites for post-translational modifications, which may play a regulatory role. By contrast, changes in RBP abundance becomes increasingly important for the RBPome responses to flg22 after 12h. Plant resistance to Pseudomonas syringae is strongly altered in mutant lines lacking individual flg22-responsive RBPs, supporting the importance of RBP dynamics in plant immunity. This study provides a comprehensive and systematic census of flg22 responsive plant RBPs, discovering novel components of plant immunity.

molecular biology↗

bioRxiv2020

The Janus-faced role of KDM5B heterogeneity in melanoma: differentiation as a situational driver of both growth arrest and drug-resistance

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. For example, melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a new dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor growth and plasticity and primes melanoma cells towards lineage-specific elimination.

cancer biology↗

bioRxiv2018

Re-annotated Nicotiana benthamiana gene models for enhanced proteomics and reverse genetics

Nicotiana benthamiana is an important model organism and representative of the Solanaceae (Nightshade) family. N. benthamiana has a complex ancient allopolyploid genome with 19 chromosomes, and an estimated genome size of 3.1Gb. Several draft assemblies of the N. benthamiana genome have been generated, however, many of the gene-models in these draft assemblies appear incorrect. Here we present a nearly non-redundant database of 42,855 improved N. benthamiana gene-models. With an estimated 97.6% completeness, the new predicted proteome is more complete than the previous proteomes. We show that the database is more sensitive and accurate in proteomics applications, while maintaining a reasonable low gene number. As a proof-of-concept we use this proteome to compare the leaf extracellular (apoplastic) proteome to a total extract of leaves. Several gene families are more abundant in the apoplast. For one of these apoplastic protein families, the subtilases, we present a phylogenetic analysis illustrating the utility of this database. Besides proteome annotation, this database will aid the research community with improved target gene selection for genome editing and off-target prediction for gene silencing.

plant biology↗