Intracellular bacterial pathogens hijack the protein machinery of infected host cells to evade their defenses and cultivate a favorable intracellular niche. The intracellular pathogen Salmonella ...enterica subsp. Typhimurium (STm) achieves this by injecting a cocktail of effector proteins into host cells that modify the activity of target host proteins. Yet, proteome-wide approaches to systematically map changes in host protein function during infection have remained challenging. Here we adapted a functional proteomics approach - Thermal-Proteome Profiling (TPP) - to systematically assess proteome-wide changes in host protein abundance and thermal stability throughout an STm infection cycle. By comparing macrophages treated with live or heat-killed STm, we observed that most host protein abundance changes occur independently of STm viability. In contrast, a large portion of host protein thermal stability changes were specific to infection with live STm. This included pronounced thermal stability changes in proteins linked to mitochondrial function (Acod1/Irg1, Cox6c, Samm50, Vdac1, and mitochondrial respiratory chain complex proteins), as well as the interferon-inducible protein with tetratricopeptide repeats, Ifit1. Integration of our TPP data with a publicly available STm-host protein-protein interaction database led us to discover that the secreted STm effector kinase, SteC, thermally destabilizes and phosphorylates the ribosomal preservation factor Serbp1. In summary, this work emphasizes the utility of measuring protein thermal stability during infection to accelerate the discovery of novel molecular interactions at the host-pathogen interface.
•First Thermal Proteome Profiling interrogation of bacterial infected host cells.•Changes in host protein thermal stability, not abundance, dominate during Salmonella infection.•Secreted SteC kinase thermally destabilises Serbp1 by mediating its phosphorylation.
The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it ...is imperative to understand how SARS‐CoV‐2 hijacks host cellular machineries on a system‐wide scale so that potential host‐directed therapies can be developed. In situ proteome‐wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS‐CoV‐2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS‐CoV‐2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS‐CoV‐2 infection.
Synopsis
Thermal proteome profiling (TPP) measurements of protein state and interaction changes in SARS‐CoV‐2 infection reveal pronounced temporal alterations in host protein thermostability during infection and indicate druggable host targets.
SARS‐CoV‐2 infection rewires the thermal stability of the host proteome.
Thermal stability changes converge on known SARS‐CoV‐2 modified pathways.
TPP informs on druggable host targets whose inhibition block SARS‐CoV‐2 replication.
Thermal proteome profiling (TPP) measurements of protein state and interaction changes in SARS‐CoV‐2 infection reveal pronounced temporal alterations in host protein thermostability during infection and indicate druggable host targets.
Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell ...lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that
Ninj1
-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.
Synopsis
Induction of ferroptosis by lipid peroxidation can lead to plasma membrane rupture, but the mediators of this rupture are unknown. This study identifies Ninjurin-1 (NINJ1) as the executor of plasma membrane permeabilization in ferroptotic macrophages and fibroblasts.
Ferroptosis induction leads to NINJ1 activation and oligomerization in macrophages and fibroblasts.
NINJ1 activation occurs downstream of lipid peroxidation and calcium influx, the first steps of ferroptosis.
NINJ1 activation triggers the last steps of ferroptosis, i.e., loss of membrane integrity and rupture.
NINJ1 controls the release of cytosolic proteins and danger-associated molecular pattern molecules from ferroptotic cells.
Ninjurin-1 (NINJ1) oligomerizes and mediates loss of plasma membrane integrity as well as plasma membrane rupture in ferroptotic cells.
Acoustic force patterning is an emerging technology that provides a platform to control the spatial location of cells in a rapid, accurate, yet contactless manner. However, very few studies have been ...reported on the usage of acoustic force patterning for the rapid arrangement of biological objects, such as cells, in a three-dimensional (3D) environment. In this study, we report on a bio-acoustic force patterning technique, which uses surface acoustic waves (SAWs) for the rapid arrangement of cells within an extracellular matrix-based hydrogel such as gelatin methacryloyl (GelMA). A proof-of-principle was achieved through both simulations and experiments based on the in-house fabricated piezoelectric SAW transducers, which enabled us to explore the effects of various parameters on the performance of the built construct. The SAWs were applied in a fashion that generated standing SAWs (SSAWs) on the substrate, the energy of which subsequently was transferred into the gel, creating a rapid, and contactless alignment of the cells (<10 s, based on the experimental conditions). Following ultraviolet radiation induced photo-crosslinking of the cell encapsulated GelMA pre-polymer solution, the patterned cardiac cells readily spread after alignment in the GelMA hydrogel and demonstrated beating activity in 5-7 days. The described acoustic force assembly method can be utilized not only to control the spatial distribution of the cells inside a 3D construct, but can also preserve the viability and functionality of the patterned cells (e.g. beating rates of cardiac cells). This platform can be potentially employed in a diverse range of applications, whether it is for tissue engineering, in vitro cell studies, or creating 3D biomimetic tissue structures.
Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein ...interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.
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•A host-pathogen protein-protein interaction map during infection•Salmonella effectors are multifunctional, with most targeting more than one host process•Effectors can converge on same host process and act in a cell-type-specific manner•SteC promotes actin bundling by phosphorylating formin-like proteins
Pathogens hijack host cells by injecting effector proteins. In this work, Walch & Selkrig et al. used quantitative proteomics to systematically map the host targets of Salmonella effectors during infection. This effort yielded hundreds of protein-protein interactions, highlighted general effector properties, and uncovered missing links in Salmonella effector biology.
Cysteine is unique among the proteinogenic amino acids due to its ability to form disulfide bonds. While this property is of vital importance for protein structures and biological processes, it ...causes difficulties for the mass spectrometric identification of cysteine-containing peptides. A common approach to overcome these problems in bottom-up proteomics is the reduction and covalent modification of sulfhydryl groups prior to enzymatic digestion. In this study, established alkylating agents and N-maleoyl amino acids with variable hydrophobicity were characterized with respect to a variety of relevant parameters and subsequently evaluated in a large-scale analysis using different ion sources. Depending on the compound, the ion source had a profound impact on the relative and absolute identification of cysteine-containing peptides. The best results were obtained by derivatization of the cysteine residues with 4-vinylpyridine and subsequent matrix-assisted laser desorption ionization (MALDI). Modification with 4-vinylpyridine increased the number of cysteine-containing peptides identified with any other compound using LC-MALDI/MS at least by a factor of 2. This experimental observation is mirrored by differences in the gas-phase basicities, which were computed for methyl thiolate derivatives of the compounds using density functional theory. With electrospray ionization (ESI), complementary use of reagents from three different compound classes, e.g., iodoacetamide, 4-vinylpyridine, and N-maleoyl beta-alanine, was beneficial compared to the application of a single reagent. Graphical abstract Cysteine-containing peptides are underrepresented in standard large scale proteomic experiments. However, deliberate matching of alkylating agent and ion source can help to increase identification rates, as outlined by characterization and validation of various reagents for cysteine-derivatization.
Following the footprints of Bill Gates, Steve Jobs and Mark Zuckerberg, there has been a misconception that students are better off quitting their studies to bring to life their ideas, create jobs ...and monetize their inventions. Having historically transitioned from manpower to mind power, we live in one of the most rapidly changing times in human history. As a result, academic institutions that are supposed to be pioneers and educators of the next generations have started to realize that they need to adapt to a new system, and change their policies to be more flexible towards patent ownership and commercialization. There is an infrastructure being developed towards students starting their own businesses while continuing with their studies. This paper aims to provide an overview of the existing landscape, the exciting rewards as well as risks awaiting a student entrepreneur, the challenges of the present ecosystem, and questions to consider prior to embarking on such a journey. Various entities influencing the start-up environment are considered, specifically for the medical technology sector. These parties include but are not limited to: scientists, clinicians, investors, academic institutions and governments. A special focus will be set on the seemingly unbridgeable gap between founding a company and a scientific career.
Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where ...individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.
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