Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Quantitative proteomics reveals unique responses to antimicrobial treatments in clinical Pseudomonas aeruginosa isolates.

IMPORTANCE: Pseudomonas aeruginosa is an important pathogen often associated with hospital-acquired infections and chronic lung infections in people with cystic fibrosis. P. aeruginosa possesses a wide array of intrinsic and adaptive mechanisms of antibiotic resistance, and the regulation of these mechanisms is complex. Label-free quantitative proteomics is a powerful tool to compare susceptible and resistant strains of bacteria and their responses to antibiotic treatments. Here we compare the proteomes of three isolates of P. aeruginosa with different antibiotic resistance profiles in response to five challenge conditions. We uncover unique and shared proteome changes for the widely used laboratory strain PAO1 and two isolates of the Liverpool epidemic strain of P. aeruginosa, LESlike1 and LESB58. Our data set provides insight into antibiotic resistance in clinically relevant Pseudomonas isolates and highlights proteins, including those with uncharacterized functions, which can be further investigated for their role in adaptive responses to antibiotic treatments.

Cross-Kingdom Infection of Macrophages Reveals Pathogen- and Immune-Specific Global Reprogramming and Adaptation.

The interactions between a host and microbe drive the health and disease status of the host. Of importance is the cause of dysbiosis in the presence of a pathogen, and critically, the relationship between the host and pathogen may evolve over time through response and adaptation. For immunocompromised individuals, dual infections are prevalent and contribute to disease severity and treatment options. Here, we explore the global reprogramming of host cells in response to immediate and established microbial infections with the human fungal pathogen Cryptococcus neoformans and the nosocomial bacterial pathogen Klebsiella pneumoniae. Using quantitative proteomics, we uncovered cross-kingdom protein-level changes associated with initial fungal infection, followed by a remarkable adaptation of the host and pathogen to a dormant state. This stabilization is disrupted over time upon bacterial infection, with the production of virulence-associated bacterial proteins and severely altered host response. We support our findings with the profiling of two major virulence determinants in C. neoformans, catalase and melanin, which demonstrate an interconnected regulation in response to both host defense and bacterial invasion. Overall, we report novel fungal and bacterial modulation of the host, including adaptation and stabilization, suggesting an opportunity to effectively treat dual infections by selectively targeting proteins critical to the host's infection stage. IMPORTANCE The relationship between the human microbiota and infectious disease outcome is a rapidly expanding area of study. Understanding how the host responds to changes in its symbiotic relationship with microbes provides new insight into how disruption can promote disease. In this study, we investigated the evolving relationship between innate immune cells of the host during immediate and established infections with fungal and bacterial pathogens, commonly observed within the lungs of immunocompromised individuals. We observed critical reprogramming of each biological system over time and in response to the changing environment, which influences microbial virulence. The goal of this important work is to improve our fundamental understanding of pathogenesis, as well as the regulatory relationships between hosts and microbes that drive disease outcome. We envision defining improved therapeutic treatment options for the host dependent on disease state to reduce the global impact and burden of infectious diseases, especially in the face of ever-increasing rates of antimicrobial resistance.

Phosphoproteomic Sample Preparation for Global Phosphorylation Profiling of a Fungal Pathogen.

Phosphorylation is a key post-translational modification central to the biological behavior of proteins. This reversible modification specifically regulates cell signaling mechanisms to control survival and growth. Moreover, microbial pathogens, including both fungi and bacteria, rely on this modification to coordinate protein production and functioning during infection and dissemination within a host. Understanding phosphorylation and its involvement with effector proteins and complex networks are now possible with the recent technological advancements of mass spectrometry. Herein, we describe a phosphopeptide enrichment strategy optimized for the invasive mycosis-causing fungal pathogen Cryptococcus neoformans. Our protocol details proper sample preparation for efficient lysis and protein extraction with minimal phosphorylation losses followed by outlined steps for enrichment, instrumentation handling, and data analysis to permit deep profiling of the global phosphoproteome. The high-throughput versatility of bottom-up proteomics combined with our sample preparation approach facilitates opportunities for in-depth phosphorylation mapping and novel biological discoveries.

Proteomic Profiling of Interplay Between Agrobacterium tumefaciens and Nicotiana benthamiana for Improved Molecular Pharming Outcomes.

Transient expression of recombinant proteins in plants is being used as a platform for production of therapeutic proteins. Benefits of this system include a reduced cost of drug development, rapid delivery of new products to the market, and an ability to provide safe and efficacious medicines for diseases. Although plant-based production systems offer excellent potential for therapeutic protein production, barriers, such as plant host defense response, exist which negatively impact the yield of product. Here we provide a protocol using tandem mass tags and mass spectrometry-based proteomics to quickly and robustly quantify the change in abundance of host defense proteins produced during the production process. These proteins can then become candidates for genetic manipulation to create host plants with reduced plant defenses capable of producing higher therapeutic protein yields.

Integrative analysis of non-small cell lung cancer patient-derived xenografts identifies distinct proteotypes associated with patient outcomes.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. Only a fraction of NSCLC harbor actionable driver mutations and there is an urgent need for patient-derived model systems that will enable the development of new targeted therapies. NSCLC and other cancers display profound proteome remodeling compared to normal tissue that is not predicted by DNA or RNA analyses. Here, we generate 137 NSCLC patient-derived xenografts (PDXs) that recapitulate the histology and molecular features of primary NSCLC. Proteome analysis of the PDX models reveals 3 adenocarcinoma and 2 squamous cell carcinoma proteotypes that are associated with different patient outcomes, protein-phosphotyrosine profiles, signatures of activated pathways and candidate targets, and in adenocarcinoma, stromal immune features. These findings portend proteome-based NSCLC classification and treatment and support the PDX resource as a viable model for the development of new targeted therapies.

MATRIX platform to analyze translation machinery remodeling in glioblastoma cells.

Here, we present a protocol using MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) platform to investigate changes of the protein synthesis machinery in U87MG glioblastoma cells in response to the rocaglate silvestrol. This protocol describes steps to perform SILAC (stable isotope labeling by amino acids in cell culture), ribosome density fractionation, protein isolation, and mass spectrometry analysis. This approach can be applied to study any adaptive remodeling of protein synthesis machineries. For complete details on the use and execution of this protocol, please refer to Ho et al. (2021).1.

Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome.

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.

Label-free quantitative proteomics identifies unique proteomes of clinical isolates of the Liverpool Epidemic Strain of Pseudomonas aeruginosa and laboratory strain PAO1.

PURPOSE: Comparative genomics and phenotypic assays have shown that antibiotic resistance profiles differ among clinical isolates of Pseudomonas aeruginosa and that genotype-phenotype associations are difficult to establish for resistance phenotypes based on these comparisons alone. EXPERIMENTAL DESIGN: Here, we used label-free quantitative proteomics to compare two isolates of the Liverpool Epidemic Strain (LES) of P. aeruginosa, LESlike1 and LESB58, and the common laboratory strain P. aeruginosa PAO1 to more accurately predict functional differences between strains. RESULTS: Our results show that the proteomes of the LES isolates are more similar to each other than to PAO1; however, a number of differences were observed in the abundance of proteins involved in quorum sensing, virulence, and antibiotic resistance, including in the comparison of LESlike1 and LESB58. Additionally, the proteomic data revealed a higher abundance of proteins involved in polymyxin and aminoglycoside resistance in LESlike1. Minimum inhibitory concentration assays showed that LESlike1 had up to 128-fold higher resistance to antibiotics from these classes. CONCLUSIONS: These findings provide an example of the ability of proteomic data to complement genotypic and phenotypic studies to understand resistance in clinical isolates. CLINICAL RELEVANCE: P. aeruginosa is a predominant pathogen in chronic lung infections in individuals with cystic fibrosis (CF). LES isolates are capable of transferring between CF patients and have been associated with increased hospital visits and antibiotic treatments.

Bacterial translation machinery for deliberate mistranslation of the genetic code.

Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.

A translational program that suppresses metabolism to shield the genome.

Translatome reprogramming is a primary determinant of protein levels during stimuli adaptation. This raises the question: what are the translatome remodelers that reprogram protein output to activate biochemical adaptations. Here, we identify a translational pathway that represses metabolism to safeguard genome integrity. A system-wide MATRIX survey identified the ancient eIF5A as a pH-regulated translation factor that responds to fermentation-induced acidosis. TMT-pulse-SILAC analysis identified several pH-dependent proteins, including the mTORC1 suppressor Tsc2 and the longevity regulator Sirt1. Sirt1 operates as a pH-sensor that deacetylates nuclear eIF5A during anaerobiosis, enabling the cytoplasmic export of eIF5A/Tsc2 mRNA complexes for translational engagement. Tsc2 induction inhibits mTORC1 to suppress cellular metabolism and prevent acidosis-induced DNA damage. Depletion of eIF5A or Tsc2 leads to metabolic re-initiation and proliferation, but at the expense of incurring substantial DNA damage. We suggest that eIF5A operates as a translatome remodeler that suppresses metabolism to shield the genome.

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment.

Mass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil-associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by P. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Murine model of ocular infection Basic Protocol 2: Murine model sample collection Basic Protocol 3: Protein extraction from eye wash Basic Protocol 4: Protein extraction from corneal tissue Basic Protocol 5: Mass spectrometry-based proteomics and bioinformatics from eye wash and corneal tissue samples.

Cancer proteome and metabolite changes linked to SHMT2.

Serine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo. Knockdown of SHMT2 expression in vitro caused a state of glycine auxotrophy and accumulation of phosphoribosylaminoimidazolecarboxamide (AICAR), an intermediate of folate/1-carbon-pathway-dependent de novo purine nucleotide synthesis. Decreased glycine in the HeLa cell-based xenograft tumors with knocked down SHMT2 was potentiated by administration of the anti-hyperglycinemia agent benzoate. However, tumor growth was not affected by SHMT2 knockdown with or without benzoate treatment. Benzoate inhibited cell proliferation in vitro, but this was independent of SHMT2 modulation. The abundance of proteins of mitochondrial respiration complexes 1 and 3 was inversely correlated with SHMT2 levels. Proximity biotinylation in vivo (BioID) identified 48 mostly mitochondrial proteins associated with SHMT2 including the mitochondrial enzymes Acyl-CoA thioesterase (ACOT2) and glutamate dehydrogenase (GLUD1) along with more than 20 proteins from mitochondrial respiration complexes 1 and 3. These data provide insights into possible mechanisms through which elevated SHMT2 in cancers may be linked to changes in metabolism and mitochondrial function.

A network of RNA-binding proteins controls translation efficiency to activate anaerobic metabolism.

Protein expression evolves under greater evolutionary constraint than mRNA levels, and translation efficiency represents a primary determinant of protein levels during stimuli adaptation. This raises the question as to the translatome remodelers that titrate protein output from mRNA populations. Here, we uncover a network of RNA-binding proteins (RBPs) that enhances the translation efficiency of glycolytic proteins in cells responding to oxygen deprivation. A system-wide proteomic survey of translational engagement identifies a family of oxygen-regulated RBPs that functions as a switch of glycolytic intensity. Tandem mass tag-pulse SILAC (TMT-pSILAC) and RNA sequencing reveals that each RBP controls a unique but overlapping portfolio of hypoxic responsive proteins. These RBPs collaborate with the hypoxic protein synthesis apparatus, operating as a translation efficiency checkpoint that integrates upstream mRNA signals to activate anaerobic metabolism. This system allows anoxia-resistant animals and mammalian cells to initiate anaerobic glycolysis and survive hypoxia. We suggest that an oxygen-sensitive RBP cluster controls anaerobic metabolism to confer hypoxia tolerance.

Supervised Learning and Mass Spectrometry Predicts the in Vivo Fate of Nanomaterials.

The surface of nanoparticles changes immediately after intravenous injection because blood proteins adsorb on the surface. How this interface changes during circulation and its impact on nanoparticle distribution within the body is not understood. Here, we developed a workflow to show that the evolution of proteins on nanoparticle surfaces predicts the biological fate of nanoparticles in vivo. This workflow involves extracting nanoparticles at multiple time points from circulation, isolating the proteins off the surface and performing proteomic mass spectrometry. The mass spectrometry protein library served as inputs, while blood clearance and organ accumulation were used as outputs to train a supervised deep neural network that predicts nanoparticle biological fate. In a double-blinded study, we tested the network by predicting nanoparticle spleen and liver accumulation with upward of 94% accuracy. Our neural network discovered that the mechanism of liver and spleen uptake is due to patterns of a multitude of nanoparticle surface adsorbed proteins. There are too many combinations to change these proteins manually using chemical or biological inhibitors to alter clearance. Therefore, we developed a technique that uses the host to act as a bioreactor to prepare nanoparticles with predictable clearance patterns that reduce liver and spleen uptake by 50% and 70%, respectively. These techniques provide opportunities to both predict nanoparticle behavior and also to engineer surface chemistries that are specifically designed by the body.

Evosep One Enables Robust Deep Proteome Coverage Using Tandem Mass Tags while Significantly Reducing Instrument Time.

The balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high-throughput manner without sacrificing in-depth proteomics coverage. We demonstrate the incorporation of Evosep One technology into our multiplexing workflow for analysis of tandem mass tag (TMT)-labeled nonsmall cell lung carcinoma (NSCLC) patient-derived xenografts (PDXs). By the use of a 30 samples per day Evosep workflow, >12 000 proteins were identified in 48 h of mass spectrometry time, which is comparable to the number of proteins identified by our conventional concatenated EASY-nLC workflow in 60 h. Shorter Evosep gradient lengths reduced the number of protein identifications by 10% while decreasing the mass analysis time by 50%. This Evosep workflow will enable quantitative analysis of multiplexed samples in less time without conceding depth of proteome coverage.

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

Synthesis of Patient-Specific Nanomaterials.

Nanoparticles are engineered from materials such as metals, polymers, and different carbon allotropes that do not exist within the body. Exposure to these exogenous compounds raises concerns surrounding toxicity, inflammation, and immune activation. These responses could potentially be mitigated by synthesizing nanoparticles directly from molecules derived from the host. However, efforts to assemble patient-derived macromolecules into structures with the same degree of size and shape tunability as their exogenous counterparts remains a significant challenge. Here we solve this problem by creating a new class of size- and shape-tunable personalized protein nanoparticles (PNP) made entirely from patient-derived proteins. PNPs are built into different sizes and shapes with the same degree of tunability as gold nanoparticles. They are biodegradable and do not activate innate or adaptive immunity following single and repeated administrations in vivo. PNPs can be further modified with specific protein cargos that remain catalytically active even after intracellular delivery in vivo. Finally, we demonstrate that PNPs created from different human patients have unique molecular fingerprints encoded directly into the structure of the nanoparticle. This new class of personalized nanomaterial has the potential to revolutionize how we treat patients and can become an integral component in the diagnostic and therapeutic toolbox.

Outer membrane lipoprotein RlpA is a novel periplasmic interaction partner of the cell division protein FtsK in Escherichia coli.

In Escherichia coli, formation of new cells is mediated by the elongasome and divisome that govern cell elongation and septation, respectively. Proper transition between these events is essential to ensure viable progeny are produced; however, the components of each complex responsible for transmission of the cell signal to shift from elongation to septation are unclear. Recently, a region within the N-terminal domain of the essential divisome protein FtsK (FtsKN) was identified that points to a key role for FtsK as a checkpoint of cell envelope remodeling during division. Here, we used site-specific in vivo UV cross-linking to probe the periplasmic loops of FtsKN for protein interaction partners critical for FtsKN function. Mass spectrometry analysis of five unique FtsKN periplasmic cross-links revealed a network of potential FtsKN interactors, one of which included the septal peptidoglycan binding protein rare lipoprotein A (RlpA). This protein was further verified as a novel interaction partner of FtsKN by an in vitro pull-down assay. Deletion of rlpA from an FtsK temperature-sensitive E. coli strain partially restored cell growth and largely suppressed cellular filamentation compared to the wild-type strain. This suggests that interaction with RlpA may be critical in suppressing septation until proper assembly of the divisome.

A feed forward loop enforces YAP/TAZ signaling during tumorigenesis.

In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway.