Biology subjects

Benz, R.

Publications and source records attributed to Benz, R..

5 recordsLinked to original sources

bioRxiv2024

Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-down Proteomics

The dynamic range challenge for detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundant proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between [~]1 {micro}g/mL and [~]10 pg/mL in absolute abundance, providing up to 105-fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 {micro}g/mL) were detected. The ability to monitor medium and some low abundant proteoforms through reproducible enrichment significantly extends the applicability of proteoform research by adding depth beyond albumin, immunoglobins and apolipoproteins to uncover many involved in immunity and cell signaling. As proteoforms carry unique information content relative to peptides, this report opens the door to deeper proteoform sequencing in clinical proteomics of disease or aging cohorts.

biochemistry↗

bioRxiv2023

Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

Bacterial AB toxins are secreted virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, they evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the resolution of the three-dimensional structure of AIP56 showed that instead of a two-domain organization, as previously predicted, AIP56 has a three-domain organization, with a NleC-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only with endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation. Considering the increasing number of putative AIP56 homologous toxins in databases, the data presented here have implications far beyond the AIP56 intoxication process and the virulence of Phdp.

microbiology↗

bioRxiv2019

Aggregatibacter actinomycetemcomitans LtxA hijacks endocytic trafficking pathways in human lymphocytes

Leukotoxin (LtxA) from oral pathogen Aggregatibacter actinomycetemcomitans is a secreted membrane-damaging protein. LtxA is internalized by {beta}2 integrin LFA-1 (CD11a/CD18) expressing leukocytes and ultimately causes cell death; however toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5 knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pH. Our results demonstrate that LtxA/LFA-1 complex gains an access to the cytosol of Jurkat cells without evidence of plasma membrane damage utilizing dynamin-dependent and clathrin-independent mechanism. Upon internalization LtxA follows the LFA-1 endocytic trafficking pathways as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp2) and CD11a. Knockdown of Rab5a resulted in loss of susceptibility of Jurkat cells to LtxA cytotoxicity suggesting that late events of LtxA endocytic trafficking are required for toxicity. The toxin trafficking via the degradation endocytic pathway may culminate in delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.

microbiology↗

bioRxiv2019

Conserved architecture of Tc toxins from human and insect pathogenic bacteria

Tc toxin complexes use a syringe-like mechanism to penetrate the membrane and translocate a toxic enzyme into the host cytosol. They are composed of three components: TcA, TcB and TcC. Until recently, low-resolution structures of TcA from different bacteria suggested that Tc toxins differ considerably in their architecture and possibly in their mechanism of action. Here, we present high-resolution structures and functional studies of five TcAs from different insect and human pathogenic bacteria. Contrary to previous expectations, their overall composition and domain organization is almost identical. The TcAs assemble as a pentamer with a central -helical channel surrounded by a shell composed of conserved -helical domains and variable {beta}-sheet domains. Essential structural features, including a conserved trefoil protein knot, are present in all five TcAs, suggesting a common mechanism of action. All TcAs form functional pores and can be combined with TcB-TcC subunits from other species resulting in chimeric holotoxins. We have identified a conserved ionic pair that stabilizes the shell, likely operating as a strong latch that only springs open after the destabilization of other regions. Our results lead to new insights into the architecture and host specificity of the Tc toxin family.

molecular biology↗

bioRxiv2019

Structure of a Tc holotoxin pore provides insights into the translocation mechanism

Tc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA) and a cocoon (TcB and TcC) encapsulating the toxic enzyme. Binding of Tcs to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. Here, we show in atomic detail an assembled Tc toxin complex from P. luminescens in the membrane. We find that the five TcA protomers conformationally adapt to fit around the cocoon during prepore-to-pore transition. The architecture of the Tc toxin complex also allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Mammalian lipids with zwitterionic head groups are preferred over other lipids for Tc toxin integration. The translocated toxic enzyme, which can be partially visualized, transiently interacts with alternating negative charges and hydrophobic stretches.

molecular biology↗