Rapid Protein Synthesis Research Articles

Altered mRNA transport and local translation in iNeurons with RNA binding protein knockdown.

Neurons rely on mRNA transport and local translation to facilitate rapid protein synthesis in processes far from the cell body. These processes allow precise spatial and temporal control of translation and are mediated by RNA binding proteins (RBPs), including those known to be associated with neurodegenerative diseases. Here, we use proteomics, transcriptomics, and microscopy to investigate the impact of RBP knockdown on mRNA transport and local translation in iPSC-derived neurons. We find thousands of transcripts enriched in neurites and that many of these transcripts are locally translated, possibly due to the shorter length of transcripts in neurites. Loss of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS)-associated RBPs TDP-43 and hnRNPA1 lead to distinct alterations in the neuritic proteome and transcriptome. TDP-43 knockdown (KD) leads to increased neuritic mRNA and translation. In contrast, hnRNPA1 leads to increased neuritic mRNA, but not translation, and more moderate effects on local mRNA profiles, possibly due to compensation by hnRNPA3. These results highlight the crucial role of FTD/ALS-associated RBPs in mRNA transport and local translation in neurons and the importance of these processes in neuron health and disease.

Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy

Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell’s viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.

A Translational Regulatory Mechanism Mediated by Hypusinated Eukaryotic Initiation Factor 5A Facilitates β-Cell Identity and Function.

As professional secretory cells, beta cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic beta cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional beta cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of beta cell identity and function. The mRNA translation function of eIF5A is only active when it is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of beta cell DHPS in mice reduces the synthesis of proteins critical to beta cell identity and function at the stage of beta cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the beta cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.

Metabolomic study on the quality differences and physiological characteristics between rice cultivated in drought and flood conditions

The differences between dry- and flood-cultivated rice and the reason behind low-quality dry-cultivated rice were clarified. The physiological traits, starch synthase activity, and grain metabolomics of ‘Longdao 18′ were measured and analyzed at four growth stages. The brown, milled, and whole-milled rice rates and AGPase, SSS, and SBE activity were lower after drought treatment than during flood cultivation, while the chalkiness, chalky grain rate, amylose (16.57–20.999%), protein (7.99–12.09%), and GBSS activity were higher. Related enzymatic gene expression showed significant differences. Metabolic results showed pyruvate, glycine, and methionine upregulation at 8DAF and higher citric, pyruvic, and α-ketoglutaric acid content at 15DAF. Therefore, 8DAF-15DAF represented the crucial quality formation period for dry-cultivated rice. At 8DAF, the respiratory pathways used amino acids as signaling molecules and alternative substrates to adapt to energy shortages, arid environments and rapid protein accumulation and synthesis. Excessive amylose synthesis at 15DAF accelerated reproductive growth, promoting rapid premature aging.

Abstract A024: A potent eIF4A1/2 inhibitor CR-1-31B down-modulates the antioxidant stress response in osteosarcoma cells and inhibits in vivo lung metastases

Abstract Background: Effective treatment of metastatic disease remains a major challenge in the improvement of patient outcomes in osteosarcoma (OS). Novel anti-metastatic therapies are needed to treat distant metastases. To this end, the current research evaluates whether targeting the dysregulated mRNA translation machinery in OS can inhibit metastases. Hypothesis: We hypothesize that mRNA translation factors present at an abnormally high abundance in OS cells support the rapid synthesis of cytoprotective proteins that are needed to survive in the oxidative stress-rich microenvironment of the lung. Experimental Approach: Databases of OS cell lines and patient tumor data (A. Sweet-Cordero, UCSF) were queried to identify mRNA translation factors with abnormal transcript levels. A limited drug screen of small molecule inhibitors (SMIs) against identified candidates was carried out to evaluate IC50 values in metastatic OS cells. A candidate inhibitor identified from these data was further characterized for synergy with chemical inducers of oxidative stress (e.g. tert-butylhydroquinone [tBHQ]) that mimics conditions encountered in the lung. Drug combination studies examined 2D and 3D tumor spheroid growth, cellular oxidative stress, and PARP-cleavage, under +/- inhibitor and +/- oxidative stress conditions. Metastatic OS cells were engineered to express an antioxidant response element (ARE)-mCherry fluorescent reporter to directly monitor the antioxidant response by fluorescence microscopy. Polysome profiling was used to assess inhibitor-mediated changes in global mRNA translation. The anti-metastatic activity of the inhibitor was tested in the ex vivo pulmonary metastasis assay (PuMA) and in in vivo metastasis models. Results: From cell and patient sample screening, eIF4A1/2 was identified as being abnormally regulated in metastatic OS cells. The SMI, CR-1-31B, specifically targets eIF4A1/2 and was found to have an IC50 of just 8 nM. CR-1-31B was found to inhibit tumor cell growth in 2D and 3D, increase cellular oxidative stress, and enhance PARP-cleavage, but only under oxidative stress conditions. Western analysis of tBHQ-treated metastatic OS cells with the ARE-mCherry reporter confirmed that the temporal expression of mCherry correlated with the upregulation of Nuclear factor erythroid 2-related factor-2 (Nrf2), a key transcriptional regulator of the antioxidant response. CR-1-31B blunted the upregulation of the antioxidant response in 2D and 3D tumor growth conditions with oxidative stress. CR-1-31B, in a dose-dependent manner, decreased the amount of polysomal mRNAs. CR-1-31B reduced the lung tumor burden in the ex vivo PuMA model, delayed primary tumor growth, and reduced lung metastases in in vivo xenograft OS models. Conclusions: Our data demonstrates that dysregulated mRNA translation is a metastatic vulnerability that can be exploited with SMIs. Altogether, these data support the inhibition of metastatic OS by CR-1-31B, highlighting the potential therapeutic utility of this selective translation inhibitor. Citation Format: Michael M. Lizardo, Christopher Hughes, Yue Zhou Huang, Alberto Delaidelli, Taras Shyp, Haifeng Zhang, Sol Snir Shaool, Poul H. Sorensen. A potent eIF4A1/2 inhibitor CR-1-31B down-modulates the antioxidant stress response in osteosarcoma cells and inhibits in vivo lung metastases [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A024.

Endoplasmic Reticulum Stress in Hepatitis B Virus and Hepatitis C Virus Infection.

Endoplasmic reticulum (ER) stress, a type of cellular stress, always occurs when unfolded or misfolded proteins accumulating in the ER exceed the protein folding capacity. Because of the demand for rapid viral protein synthesis after viral infection, viral infections become a risk factor for ER stress. The hepatocyte is a cell with large and well-developed ER, and hepatitis virus infection is widespread in the population, indicating the interaction between hepatitis viruses and ER stress may have significance for managing liver diseases. In this paper, we review the process that is initiated by the hepatocyte through ER stress against HBV and HCV infection and explain how this information can be helpful in the treatment of HBV/HCV-related diseases.

Maintenance of tRNA and elongation factors supports T3SS proteins translational elongations in pathogenic bacteria during nutrient starvation

BackgroundSufficient nutrition contributes to rapid translational elongation and protein synthesis in eukaryotic cells and prokaryotic bacteria. Fast synthesis and accumulation of type III secretion system (T3SS) proteins conduce to the invasion of pathogenic bacteria into the host cells. However, the translational elongation patterns of T3SS proteins in pathogenic bacteria under T3SS-inducing conditions remain unclear. Here, we report a mechanism of translational elongation of T3SS regulators, effectors and structural protein in four model pathogenic bacteria (Pseudomonas syringae, Pseudomonas aeruginosa, Xanthomonas oryzae and Ralstonia solanacearum) and a clinical isolate (Pseudomonas aeruginosa UCBPP-PA14) under nutrient-limiting conditions. We proposed a luminescence reporter system to quantitatively determine the translational elongation rates (ERs) of T3SS regulators, effectors and structural protein under different nutrient-limiting conditions and culture durations.ResultsThe translational ERs of T3SS regulators, effectors and structural protein in these pathogenic bacteria were negatively regulated by the nutrient concentration and culture duration. The translational ERs in 0.5× T3SS-inducing medium were the highest of all tested media. In 1× T3SS-inducing medium, the translational ERs were highest at 0 min and then rapidly decreased. The translational ERs of T3SS regulators, effectors and structural protein were inhibited by tRNA degradation and by reduced levels of elongation factors (EFs).ConclusionsRapid translational ER and synthesis of T3SS protein need adequate tRNAs and EFs in nutrient-limiting conditions. Numeric presentation of T3SS translation visually indicates the invasion of bacteria and provides new insights into T3SS expression that can be applied to other pathogenic bacteria.

Superfast desulfurization for protein chemical synthesis and modification

Superfast desulfurization for protein chemical synthesis and modification

Correlation Analysis between Sports and Antiaging Based on Medical Big Data

With the aging of the population in China and even in the world becoming more and more serious, China has become the country with the largest proportion of the elderly population, and a series of social problems such as health and medical care brought about by aging are being actively responded by policies. Aging has also become a natural law that human beings cannot break free from. Although exercise cannot reverse the aging process, it can weaken the adverse effects caused by aging. Having good physical quality, keeping a happy mood, comfortable living environment, and friendly social relations are the secret recipe for prolonging life and resisting cell aging. On the contrary, if physical and mental exhaustion, various chronic diseases, negative life events, bad living environment, and social relations will seriously affect human life and quality of life. Sports promote the metabolism of the whole body. Exercise can stabilize blood sugar, avoid cardiovascular diseases, and even get a good mood. At the cellular level, exercise facilitates the transmission and absorption of nutrients, thus making tissues healthier to cope with the stress of daily life. According to the statistics of loving sports and not loving sports through medical big data, the physical fitness and cell health of the elderly who love exercise every day are better than those who do not love sports. Sports are controlled by the central nervous system, and there is a correlation between changes in motor ability and cognitive impairment. Aging is accompanied by the deterioration of skeletal muscle quality and strength, which is mainly due to the rapid degradation and slow synthesis of protein in skeletal muscle. Therefore, exercise is one of the most effective ways to delay the aging of muscles and bones. According to the great potential value of medical big data, this paper analyzes and explains the correlation between sports and antiaging.

Protein Kinase Signaling by Shiga Toxin Subunits.

Background:Escherichia coli produces Shiga toxin (Stx), a pentamer composed of one A subunit and four B subunits. The B subunit of Stx (StxB) mediated the attachment of the holotoxin to the cell surface while the A subunit (StxA) has N-glycosidase activity, resulting in protein synthesis and cell death inhibition. Stx-induced cytotoxicity and apoptosis have been observed in various cell lines, although the signaling effectors are not precisely defined. Activated by protein kinases (PK), the signaling pathway in human tumors plays an oncogenic role. Tumor proliferation, survival, and metastasis are promoted by kinase receptors. In this regard, PK regulatory effects on the cellular constituents of the tumor microenvironment can affect immunosuppressive purposes.Methods:In this study, kinase inhibitors were used to evaluate the influence of Stx and its subunits on HeLa and Vero cells. Selective inhibitors of protein kinase C (PKC), CaM kinase (calmodulin kinase), protein kinase A (PKA), and protein kinase G (PKG) were used to compare the signaling activity of each subunit.Results:The ribotoxic activity in the target cells will lead to rapid protein synthesis inhibition and cell death in the mammalian host. The expression of Bcl2 family members was also assessed. Protein kinase signaling by Stx and its A and B subunits was induced by PKA, PKG, and PKC in HeLa cells. CaM kinase induction was significant in Vero cells. StxB significantly induced the pro-apoptotic Bax signaling factor in HeLa cells.Conclusion:The assessment of different signaling pathways utilized by Stx and its subunits could help in a better understanding of various cell death responses. The use of inhibitors can block cell damage and disease progression and create therapeutic compounds for targeted cancer therapy. Inhibition of these pathways is the primary clinical goal.

Rapid Chemical Synthesis of Self-Assembling Semi-Synthetic Proteins.

The design of well-defined monodispersed self-assembling semi-synthetic proteins is emerging as a promising research avenue. These proteins hold great potential to be used as scaffolds for various protein nanotechnology applications. Currently, there are very few chemical methods reported; however, they suffer from elaborate multistep organic synthesis. Herein, we report a new chemical methodology for the rapid synthesis of a diverse set of semi-synthetic protein families, which include protein amphiphiles, facially amphiphilic protein-dendron conjugates, and pH-sensitive protein-dendron conjugates. This chemical method holds great potential to access a wide variety of semi-synthetic proteins in a short time.

Analysis of the Innovation Trend in Cell-Free Synthetic Biology.

Cell-free synthetic biology is a maturing field that aims to assemble biomolecular reactions outside cells for compelling applications in drug discovery, metabolic engineering, biomanufacturing, diagnostics, and education. Cell-free systems have several key features. They circumvent mechanisms that have evolved to facilitate species survival, bypass limitations on molecular transport across the cell wall, enable high-yielding and rapid synthesis of proteins without creating recombinant cells, and provide high tolerance towards toxic substrates or products. Here, we analyze ~750 published patents and ~2000 peer-reviewed manuscripts in the field of cell-free systems. Three hallmarks emerged. First, we found that both patent filings and manuscript publications per year are significantly increasing (five-fold and 1.5-fold over the last decade, respectively). Second, we observed that the innovation landscape has changed. Patent applications were dominated by Japan in the early 2000s before shifting to China and the USA in recent years. Finally, we discovered an increasing prevalence of biotechnology companies using cell-free systems. Our analysis has broad implications on the future development of cell-free synthetic biology for commercial and industrial applications.

Impaired eIF5A function causes a Mendelian disorder that is partially rescued in model systems by spermidine

The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes.

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.

Rapid synthesis of dual proteins co-functionalized gold nanoclusters for ratiometric fluorescence sensing of polynucleotide kinase activity

Herein, we reported a facile strategy for rapidly synthesizing gold nanoclusters co-functionalized by dual proteins (bovine serum albumin and lysozyme, BL-AuNCs). The BL-AuNCs exhibited intense fluorescence emission at 640 nm and good biochemical stability. And a sensitive and simple ratio fluorescent method was constructed for polynucleotide kinase (PNK) activity analysis by utilizing BL-AuNCs and dsDNA-intercalating dye-SYBR Green I (SGI) as sensing receptors for the first time. BL-AuNCs were modified with specific designed double-stranded DNA (dsDNA) (BL-AuNCs-dsDNA). SGI, a specific intercalator of dsDNA, displayed strong fluorescence emission at 526 nm when intercalated into the dsDNA on BL-AuNCs. With adenosine triphosphate (ATP), PNK can catalyze phosphorylation of dsDNA at 5′-terminus. Phosphorylated dsDNA was specially recognized and degraded by λ exonuclease (λ exo) to produce single-stranded DNA and mononucleotides, which lacked ability to combine with SGI. Therefore, the fluorescent signal at 526 nm would decrease, while the BL-AuNCs fluorescence at 640 nm kept unaffected and was employed as the reference signal. The PNK activity was detected by monitoring variation of the ratio of fluorescence intensity (F526/F640) with a low detection limit of 0.012 U mL−1. This ratio fluorescent strategy was also applied for PNK determination in cell lysates with satisfying results.

Microenvironmental Hypoxia Induces Dynamic Changes in Lung Cancer Synthesis and Secretion of Extracellular Vesicles.

Simple SummaryHuman cells can communicate with each other by releasing small packets of protein called ‘vesicles’ that are absorbed by other cells nearby and at distant locations, leading to major changes in biological activity. This is also the case for cancer cells within developing tumours, which become starved of oxygen when they outgrow local blood supplies. We wondered whether oxygen starvation might cause cancer cells to alter the protein cargo they package into vesicles, which could play an important role in supporting further tumour development. We found that under oxygen-starved conditions, cancer cells release far higher numbers of vesicles that are actively packed with proteins known to enhance tumour survival, support distribution to other body sites, and suppress patient immune responses. Cancer vesicles could therefore be used to develop new diagnostic tests that inform doctors about disease progression, and may also represent useful drug targets for new types of patient treatment.Extracellular vesicles (EVs) mediate critical intercellular communication within healthy tissues, but are also exploited by tumour cells to promote angiogenesis, metastasis, and host immunosuppression under hypoxic stress. We hypothesize that hypoxic tumours synthesize hypoxia-sensitive proteins for packing into EVs to modulate their microenvironment for cancer progression. In the current report, we employed a heavy isotope pulse/trace quantitative proteomic approach to study hypoxia sensitive proteins in tumour-derived EVs protein. The results revealed that hypoxia stimulated cells to synthesize EVs proteins involved in enhancing tumour cell proliferation (NRSN2, WISP2, SPRX1, LCK), metastasis (GOLM1, STC1, MGAT5B), stemness (STC1, TMEM59), angiogenesis (ANGPTL4), and suppressing host immunity (CD70). In addition, functional clustering analyses revealed that tumour hypoxia was strongly associated with rapid synthesis and EV loading of lysosome-related hydrolases and membrane-trafficking proteins to enhance EVs secretion. Moreover, lung cancer-derived EVs were also enriched in signalling molecules capable of inducing epithelial-mesenchymal transition in recipient cancer cells to promote their migration and invasion. Together, these data indicate that lung-cancer-derived EVs can act as paracrine/autocrine mediators of tumorigenesis and metastasis in hypoxic microenvironments. Tumour EVs may, therefore, offer novel opportunities for useful biomarkers discovery and therapeutic targeting of different cancer types and at different stages according to microenvironmental conditions.

Network theory of the bacterial ribosome.

In the past two decades, research into the biochemical, biophysical and structural properties of the ribosome have revealed many different steps of protein translation. Nevertheless, a complete understanding of how they lead to a rapid and accurate protein synthesis still remains a challenge. Here we consider a coarse network analysis in the bacterial ribosome formed by the connectivity between ribosomal (r) proteins and RNAs at different stages in the elongation cycle. The ribosomal networks are found to be dis-assortative and small world, implying that the structure allows for an efficient exchange of information between distant locations. An analysis of centrality shows that the second and fifth domains of 23S rRNA are the most important elements in all of the networks. Ribosomal protein hubs connect to much fewer nodes but are shown to provide important connectivity within the network (high closeness centrality). A modularity analysis reveals some of the different functional communities, indicating some known and some new possible communication pathways Our mathematical results confirm important communication pathways that have been discussed in previous research, thus verifying the use of this technique for representing the ribosome, and also reveal new insights into the collective function of ribosomal elements.

Effective Method for Accurate and Sensitive Quantitation of Rapid Changes of Newly Synthesized Proteins.

Protein synthesis is quickly and tightly regulated in cells to adapt to the ever-changing extracellular and intracellular environment. Accurate quantitation of rapid protein synthesis changes can provide insights into protein functions and cellular activities, but it is very challenging to achieve because of the lack of effective analysis methods. Here, we developed an effective mass spectrometry-based method named quantitative O-propargyl-puromycin tagging (QOT) by integrating O-propargyl-puromycin (OPP) labeling, bioorthogonal chemistry, and multiplexed proteomics for global and quantitative analysis of rapid protein synthesis. The current method enables us to accurately quantitate rapid changes of newly synthesized proteins because, unlike amino acids and their analogs, OPP can be utilized by the ribosome immediately without being activated and conjugated to tRNA, and thus cell starvation or pretreatment is not required. This method was applied to quantitate rapid changes of protein synthesis in THP-1 macrophages treated with lipopolysaccharide (LPS). For 15-min labeling, >3000 proteins were quantitated, and the synthesis of 238 proteins was significantly altered, including transcription factors and cytokines. The results demonstrated that protein synthesis was modulated to facilitate protein secretion in macrophages in response to LPS. Considering the importance of protein synthesis, this method can be extensively applied to investigate rapid changes of protein synthesis in the biological and biomedical research fields.

Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection.

SummaryThe interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined click-chemistry with pulsed stable isotope labeling of amino acids in cell culture (pSILAC-AHA) to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen, Salmonella enterica Typhimurium (STm). We monitored newly synthesised proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified active cathepsins re-traffic to the nucleus and are linked to cell death. Pharmacological cathepsin inhibition and nuclear-targeting of a cellular cathepsin inhibitor (Stefin B) suppressed STm-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that LPS transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced Gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolving host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Molecular Biology of Escherichia Coli Shiga Toxins' Effects on Mammalian Cells.

Shiga toxins (Stxs), syn. Vero(cyto)toxins, are potent bacterial exotoxins and the principal virulence factor of enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin-producing E. coli (STEC). EHEC strains, e.g., strains of serovars O157:H7 and O104:H4, may cause individual cases as well as large outbreaks of life-threatening diseases in humans. Stxs primarily exert a ribotoxic activity in the eukaryotic target cells of the mammalian host resulting in rapid protein synthesis inhibition and cell death. Damage of endothelial cells in the kidneys and the central nervous system by Stxs is central in the pathogenesis of hemolytic uremic syndrome (HUS) in humans and edema disease in pigs. Probably even more important, the toxins also are capable of modulating a plethora of essential cellular functions, which eventually disturb intercellular communication. The review aims at providing a comprehensive overview of the current knowledge of the time course and the consecutive steps of Stx/cell interactions at the molecular level. Intervention measures deduced from an in-depth understanding of this molecular interplay may foster our basic understanding of cellular biology and microbial pathogenesis and pave the way to the creation of host-directed active compounds to mitigate the pathological conditions of STEC infections in the mammalian body.