Shiga Toxins as Antitumor Tools

Shiga toxins (Stxs), also known as Shiga-like toxins (SLT) or verotoxins (VT), constitute a family of structurally and functionally related cytotoxic proteins produced by the enteric pathogens Shigella dysenteriae type 1 and Stx-producing Escherichia coli (STEC). Infection with these bacteria causes bloody diarrhea and other pathological manifestations that can lead to HUS (hemolytic and uremic syndrome). At the cellular level, Stxs bind to the cellular receptor Gb3 and inhibit protein synthesis by removing an adenine from the 28S rRNA. This triggers multiple cellular signaling pathways, including the ribotoxic stress response (RSR), unfolded protein response (UPR), autophagy and apoptosis. Stxs cause several pathologies of major public health concern, but their specific targeting of host cells and efficient delivery to the cytosol could potentially be exploited for biomedical purposes. Moreover, high levels of expression have been reported for the Stxs receptor, Gb3/CD77, in Burkitt’s lymphoma (BL) cells and on various types of solid tumors. These properties have led to many attempts to develop Stxs as tools for biomedical applications, such as cancer treatment or imaging, and several engineered Stxs are currently being tested. We provide here an overview of these studies.

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ScreeningMycobacterium tuberculosisSecreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector

mSphere ◽

10.1128/msphere.00354-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 2

Author(s):

Chelsea E. Stamm ◽

Breanna L. Pasko ◽

Sujittra Chaisavaneeyakorn ◽

Luis H. Franco ◽

Vidhya R. Nair ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mycobacterium Tuberculosis ◽

Unfolded Protein Response ◽

Molecular Mechanisms ◽

Membrane Binding ◽

Host Cells ◽

Secreted Proteins ◽

Content Type ◽

Unfolded Protein ◽

Protein Response

ABSTRACTMycobacterium tuberculosis(Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCEAdvances have been made to identify secreted proteins ofMycobacterium tuberculosisduring animal infections. These data, combined with transposon screens identifying genes important forM. tuberculosisvirulence, have generated a vast resource of potentialM. tuberculosisvirulence proteins. However, the function of many of these proteins inM. tuberculosispathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200M. tuberculosissecreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) duringM. tuberculosisinfection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

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Modulation of SARS-CoV-2 Spike-induced Unfolded Protein Response (UPR) in HEK293T cells by selected small chemical molecules

10.1101/2021.02.04.429769 ◽

2021 ◽

Author(s):

Bijina Balakrishnan ◽

Kent Lai

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

Viral Infections ◽

Host Cells ◽

Transfected Cells ◽

Unfolded Protein ◽

Infected Cells ◽

Protein Response ◽

Small Chemical ◽

Stress Activation

Coronaviruses (CoV) exploits the endoplasmic reticulum (ER) of the host cells for replication and in doing so, increases ER stress. evokes Unfolded Protein Response (UPR) and possibly autophagy, which could all attribute to the pathophysiology of the viral infections. To date, little is known about the roles of ER stress, UPR, and autophagy in SARS-CoV-2 infection. Here we over-expressed the viral Spike (S) protein in cultured HEK293T cells, as it has been shown that such protein is largely responsible for UPR activation in other CoV-infected cells. We noticed, in the transfected cells, heightened ER stress, activation of the PERK-eIF2α arm of the UPR, induction of autophagy and cell death. When we treated the transfected cells with Tauroursodeoxycholic acid (TUDCA), 4-phenyl butyric acid (PBA), Salubrinal, Trazadone hydrochloride, and Dibenzoylmethane (DBM), we saw reduced the BiP/GRP78 levels, but only PBA and TUDCA could significantly diminish the levels of peIF2α and autophagy expression.

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High-content screening image dataset and quantitative image analysis of Salmonella infected human cells

BMC Research Notes ◽

10.1186/s13104-019-4844-5 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

Antony N. Antoniou ◽

Simon J. Powis ◽

Janos Kriston-Vizi

Keyword(s):

Endoplasmic Reticulum ◽

Image Analysis ◽

Unfolded Protein Response ◽

Confocal Imaging ◽

Host Cells ◽

High Content Screening ◽

Microscopic Images ◽

Unfolded Protein ◽

Confocal Fluorescence ◽

Protein Response

Abstract Objectives Salmonella bacteria can induce the unfolded protein response, a cellular stress response to misfolding proteins within the endoplasmic reticulum. Salmonella can exploit the host unfolded protein response leading to enhanced bacterial replication which was in part mediated by the induction and/or enhanced endo-reticular membrane synthesis. We therefore wanted to establish a quantitative confocal imaging assay to measure endo-reticular membrane expansion following Salmonella infections of host cells. Data description High-content screening confocal fluorescence microscopic image set of Salmonella infected HeLa cells is presented. The images were collected with a PerkinElmer Opera LX high-content screening system in seven 96-well plates, 50 field-of-views and DAPI, endoplasmic reticulum tracker channels and Salmonella mCherry protein in each well. Totally 93,300 confocal fluorescence microscopic images were published in this dataset. An ImageJ high-content image analysis workflow was used to extract features. Cells were classified as infected and non-infected, the mean intensity of endoplasmic reticulum tracker under Salmonella bacteria was calculated. Statistical analysis was performed by an R script, quantifying infected and non-infected cells for wild-type and ΔsifA mutant cells. The dataset can be further used by researchers working with big data of endoplasmic reticulum fluorescence microscopic images, Salmonella bacterial infection images and human cancer cells.

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Pseudorabies Virus Infection Induces Endoplasmic Reticulum Stress Through PERK and IRE1 Pathways of Unfolded Protein Response

10.21203/rs.3.rs-1087342/v1 ◽

2021 ◽

Author(s):

Li Chen ◽

Minshu Ni ◽

Waqas Ahmed ◽

Yue Xu ◽

Xi Bao ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Unfolded Protein Response ◽

Pseudorabies Virus ◽

Viral Infections ◽

Host Cells ◽

Unfolded Protein ◽

Xbp1 Mrna ◽

Enhanced Expression ◽

Protein Response

Abstract Pseudorabies virus (PRV) is a pathogen of swine resulting in devastating disease. Some viral infections can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) to restore ER homeostasis. However, the mechanism of how PRV induces ER stress and UPR activation remains unclear. Here, levels of proteins or transcriptional factors of three UPR pathways were examined in suspension-cultured BHK-21 cells to investigate PRV-induced ER stress. Results showed that PRV triggered ER stress and UPR of the host cells with the upregulated expression of glucose-related protein 78 kD and 94 kD (GRP78 and GRP94). The protein kinase RNA-like ER kinase (PERK) pathway was activated to upregulate ATF4, CHOP, and GADD34 expression. Additionally, the inositol requiring kinase 1 (IRE1) pathway was triggered by splicing of X box-binding protein 1 (XBP1) mRNA and the enhanced expression of p58IPK and EDEM1. Furthermore, our data demonstrated that PRV took advantage of ER stress to accelerate its replication with the activation of the PERK and IRE1 pathways in suspension-cultured BHK-21 cells, and the glycoprotein B played a crucial role in ER stress.

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ATF6-mediated unfolded protein response facilitates AAV2 transduction by releasing the suppression of AAV receptor on ER stress

Journal of Virology ◽

10.1128/jvi.01103-21 ◽

2021 ◽

Author(s):

Mengtian Cui ◽

Qingfang Zhao ◽

Jing Wang ◽

Yang Si ◽

Shan Cheng ◽

...

Keyword(s):

Gene Therapy ◽

Er Stress ◽

Unfolded Protein Response ◽

Viral Vector ◽

Host Cells ◽

Transduction Efficiency ◽

Unfolded Protein ◽

Physiological Properties ◽

Inhibitory Molecule ◽

Protein Response

Adeno-associated virus (AAV) is extensively used as a viral vector to deliver therapeutic genes during human gene therapy. A high affinity cellular receptor (AAVR) for most serotypes was recently identified, however, its biological function as a gene product remains unclear. In this study, we used AAVR knockdown cell models to show that AAVR depletion significantly attenuated cells to activate unfolded protein response (UPR) pathways, when exposed to the endoplasmic reticulum (ER) stress inducer, tunicamycin. By analyzing three major UPR pathways, we found that ATF6 signaling was most affected in an AAVR-dependent fashion, distinct to CHOP and XBP1 branches. AAVR capacity in UPR regulation required the full native AAVR protein, and AAV2 capsid binding to the receptor altered ATF6 dynamics. Conversely, the transduction efficiency of AAV2 was associated with changes in ATF6 signaling in host cells following treatment with different small molecules. Thus, AAVR served as an inhibitory molecule to repress UPR responses via a specificity for ATF6 signaling, and the AAV2 infection route involved the release from AAVR-mediated ATF6 repression, thereby facilitating viral intracellular trafficking and transduction. Importance The native function of the AAVR as an ER-Golgi localized protein is largely unknown. We showed that AAVR acted as a functional molecule to regulate UPR signaling under induced ER stress. AAVR inhibited the activation of the transcription factor, ATF6, whereas receptor binding to AAV2 released the suppression effects. This finding has expanded our understanding of AAV infection biology in terms of the physiological properties of AAVR in host cells. Importantly, our research provides a possible strategy which may improve the efficiency of AAV mediated gene delivery during gene therapy.

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Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication

Journal of General Virology ◽

10.1099/vir.0.057265-0 ◽

2014 ◽

Vol 95 (1) ◽

pp. 71-79 ◽

Cited By ~ 37

Author(s):

Sankar Bhattacharyya ◽

Utsav Sen ◽

Sudhanshu Vrati

Keyword(s):

Japanese Encephalitis Virus ◽

Unfolded Protein Response ◽

Japanese Encephalitis ◽

Mammalian Cells ◽

Cellular Response ◽

Encephalitis Virus ◽

Host Cells ◽

Rnase Activity ◽

Unfolded Protein ◽

Protein Response

Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.

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Sensing of Bacterial Type IV Secretion via the Unfolded Protein Response

mBio ◽

10.1128/mbio.00418-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 60

Author(s):

Maarten F. de Jong ◽

Tregei Starr ◽

Maria G. Winter ◽

Andreas B. den Hartigh ◽

Robert Child ◽

...

Keyword(s):

Unfolded Protein Response ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Content Type ◽

Unfolded Protein ◽

Type Iv ◽

Er Chaperone ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACTHost cytokine responses toBrucella abortusinfection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces proinflammatory responses. Ectopically expressed VceC interacted with the endoplasmic reticulum (ER) chaperone BiP/Grp78 and localized to the ER of HeLa cells. ER localization of VceC required a transmembrane domain in its N terminus. Notably, the expression of VceC resulted in reorganization of ER structures. In macrophages, VceC was required forB. abortus-induced inflammation by induction of the unfolded protein response by a process requiring inositol-requiring transmembrane kinase/endonuclease 1. Altogether, these findings suggest that translocation of the T4SS effector VceC induces ER stress, which results in the induction of proinflammatory host cell responses duringB. abortusinfection.IMPORTANCEBrucellaspecies are pathogens that require a type IV secretion system (T4SS) to survive in host cells and to maintain chronic infection. By as-yet-unknown pathways, the T4SS also elicits inflammatory responses in infected cells. Here we show that inflammation caused by the T4SS results in part from the sensing of a T4SS substrate, VceC, that localizes to the endoplasmic reticulum (ER), an intracellular site ofBrucellareplication. Possibly via binding of the ER chaperone BiP, VceC causes ER stress with concomitant expression of proinflammatory cytokines. Thus, induction of the unfolded protein response may represent a novel pathway by which host cells can detect pathogens deploying a T4SS.

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The Impacts of Unfolded Protein Response in the Retinal Cells During Diabetes: Possible Implications on Diabetic Retinopathy Development

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.615125 ◽

2021 ◽

Vol 14 ◽

Author(s):

Kaiser Alam ◽

Yusuf Akhter

Keyword(s):

Diabetic Retinopathy ◽

Unfolded Protein Response ◽

Clinical Management ◽

Close Association ◽

Cellular Level ◽

Eye Disease ◽

Unfolded Protein ◽

Working Age ◽

Diabetic Population ◽

Protein Response

Diabetic retinopathy (DR) is a vision-threatening, chronic, and challenging eye disease in the diabetic population. Despite recent advancements in the clinical management of diabetes, DR remains the major cause of blindness in working-age adults. A better understanding of the molecular and cellular basis of DR development will aid in identifying therapeutic targets. Emerging pieces of evidence from recent research in the field of ER stress have demonstrated a close association between unfolded protein response (UPR)-associated cellular activities and DR development. In this minireview article, we shall provide an emerging understating of how UPR influences DR pathogenesis at the cellular level.

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Arms Race between Enveloped Viruses and the Host ERAD Machinery

10.20944/preprints201608.0043.v1 ◽

2016 ◽

Author(s):

Dylan A. Frabutt ◽

Yong-Hui Zheng

Keyword(s):

Cellular Localization ◽

Viral Envelope ◽

Host Cells ◽

Envelope Glycoproteins ◽

Viral Fusion ◽

Enveloped Viruses ◽

Protein Levels ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

Enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. These viruses express envelope glycoproteins that are singularly important during infection of host cells by mediating fusion between the viral envelope and host cell membranes. Despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. Their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (ER). However, viral protein expression can cause stress in the ER, and host cells respond to alleviate the ER stress in the form of the unfolded protein response (UPR); the effects of which have been observed potentiating or inhibiting viral infection. One important arm of UPR is to elevate the capacity of the ER-associated protein degradation (ERAD) pathway, which is comprised of host quality control machinery that ensures proper protein folding. In this review, we provide relevant details regarding viral envelope glycoproteins, UPR, ERAD, and their interactions in host cells.

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Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells

10.1101/2020.04.14.042069 ◽

2020 ◽

Author(s):

Leonardo Augusto ◽

Jennifer Martynowicz ◽

Parth H. Amin ◽

Nada S. Alakhras ◽

Mark H. Kaplan ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

The Body ◽

Host Cells ◽

Migratory Activity ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

AbstractToxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host cells, which facilitates dissemination. Here we show that Toxoplasma triggers the unfolded protein response (UPR) in host cells through calcium release from the endoplasmic reticulum (ER). We further found that host IRE1, an ER stress sensor protein activated during Toxoplasma infection, also plays a noncanonical role in actin remodeling by binding filamin A in infected cells. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo. Our study identifies novel mechanisms used by Toxoplasma to induce dissemination of infected cells, providing new insights into strategies for treatment of toxoplasmosis.ImportanceCells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this study, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration does not involve the enzymatic activity of IRE1, but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo. Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body.

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