scholarly journals Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

2019 ◽  
Author(s):  
Tomasz Janiszewski ◽  
Sonia Kołt ◽  
Dion Kaiserman ◽  
Scott Snipas ◽  
Shuang Li ◽  
...  
Keyword(s):  
Amino Acids ◽  
Target Cell ◽  
Unnatural Amino Acids ◽  
Granzyme B ◽  
Target Cells ◽  
Substrate Preferences ◽  
Depth Analysis ◽  
Fluorogenic Probes ◽  
Key Aspects ◽  
And Function

AbstractDespite many studies on the cytotoxic protease granzyme B, key aspects of its function remain unexplored due to the lack of selective probes for its activity. In this study, we fully mapped the substrate preferences of GrB using a set of unnatural amino acids, demonstrating previously unknown GrB substrate preferences that we then used to design novel substrate-based inhibitors and a GrB-activatable activity-based probe. We showed that our GrB probes react poorly with caspases, making them ideal for the in-depth analysis of GrB localization and function in cells. With our quenched fluorescence substrate, we determined GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with the GrB attack MDA-MB-231 target cells, and active GrB influences its target cell killing efficiency.

scholarly journals Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes

2020 ◽  
Vol 295 (28) ◽  
pp. 9567-9582
Author(s):  
Tomasz Janiszewski ◽  
Sonia Kołt ◽  
Dion Kaiserman ◽  
Scott J. Snipas ◽  
Shuang Li ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Unnatural Amino Acids ◽  
Viral Infections ◽  
Granzyme B ◽  
Target Cells ◽  
Fluorescent Substrate ◽  
Substrate Preferences ◽  
Key Aspects ◽  
And Function

Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.

scholarly journals NOVEL IMMUNOTHERAPEUTIC TARGETED GRANZYME DELIVERY SYSTEMS IN TREATMENT OF MALIGNANT TUMORS

2021 ◽  
Vol 20 (2) ◽  
pp. 31-41
Author(s):  
I. V. Yarosh ◽  
V. A. Misyurin ◽  
I. I. Krasnyuk
Keyword(s):  
Cell Death ◽  
Target Cell ◽  
Serine Proteases ◽  
Malignant Tumors ◽  
Granzyme B ◽  
Killer Cell ◽  
Cascade Reactions ◽  
Target Cells ◽  
Cell Property ◽  
Infected Cells

Cytotoxicity is the main human killer cell property. The cytotoxicity reaction of human killer cells is achieved through a complex of molecules, including perforins, granzyme, cathepsin and others. However, only one molecule is enough for target cell death: granzyme. Other molecules are intended for granzyme activation and its delivery to the target cell cytoplasm. Granzymes are a whole family of serine proteases that perform their function in the human body as integral cytolytic effectors during programmed cell death of cancer and pathogen-infected cells. Secreted mainly by cytotoxic T-lymphocytes and NK-cells, granzymes initiate apoptosis via caspase-dependent and caspase-independent pathways. These natural properties make granzymes one of the most promising human enzymes for use in the development of targeted therapeutic strategies in the treatment of various types of cancer.The most promising is granzyme B, because it has the most powerful effector properties. Due to the initiation of cascade reactions that activate apoptosis, granzyme is attractive as a basis for the development of medicines applicable in clinical oncology. At this time, several approaches have been developed for delivering granzyme molecules to tumor cells and facilitating its penetration through the cell membrane. Moreover, some solutions are proposed to overcome the resistance of target cells to granzyme-mediated apoptosis. These approaches are discussed in this review.The purpose of this review was to systematize information on the use of granzyme B as a nanostructured drug delivery system in the treatment of solid and hematological malignancies. In addition, this review discusses ways to overcome the resistance of granzyme penetration into target cells.

scholarly journals Glia-derived exosomal miR-274 targets Sprouty in trachea and synaptic boutons to modulate growth and responses to hypoxia

2019 ◽  
Vol 116 (49) ◽  
pp. 24651-24661 ◽  
Author(s):  
Yi-Wei Tsai ◽  
Hsin-Ho Sung ◽  
Jian-Chiuan Li ◽  
Chun-Yen Yeh ◽  
Pei-Yi Chen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Target Cell ◽  
Target Gene ◽  
Mapk Signaling ◽  
Target Cells ◽  
Null Mutant ◽  
Specific Expression ◽  
Exosome Biogenesis ◽  
Synaptic Boutons ◽  
And Function

Secreted exosomal microRNAs (miRNAs) mediate interorgan/tissue communications by modulating target gene expression, thereby regulating developmental and physiological functions. However, the source, route, and function in target cells have not been formally established for specific miRNAs. Here, we show that glial miR-274 non-cell-autonomously modulates the growth of synaptic boutons and tracheal branches. Whereas the precursor form of miR-274 is expressed in glia, the mature form of miR-274 distributes broadly, including in synaptic boutons, muscle cells, and tracheal cells. Mature miR-274 is secreted from glia to the circulating hemolymph as an exosomal cargo, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. We further show that miR-274 can function in the neurons or tracheal cells to modulate the growth of synaptic boutons and tracheal branches, respectively. Also, miR-274 uptake into the target cells by AP-2–dependent mechanisms modulates target cell growth. In the target cells, miR-274 down-regulates Sprouty (Sty) through a targeting sequence at the sty 3′ untranslated region, thereby enhancing MAPK signaling and promoting cell growth. miR-274 expressed in glia of an mir-274 null mutant is released as an exosomal cargo in the circulating hemolymph, and such glial-specific expression resets normal levels of Sty and MAPK signaling and modulates target cell growth. mir-274 mutant larvae are hypersensitive to hypoxia, which is suppressed by miR-274 expression in glia or by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.

Entry and Trafficking of Granzyme B in Target Cells During Granzyme B-Perforin–Mediated Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1044-1054 ◽  
Author(s):  
Michael J. Pinkoski ◽  
Marita Hobman ◽  
Jeffrey A. Heibein ◽  
Kevin Tomaselli ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Indirect Immunofluorescence ◽  
Target Cell ◽  
Granzyme B ◽  
Fluorescein Isothiocyanate ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Direct Visualization ◽  
New Model ◽  
American Society

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin–induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death. © 1998 by The American Society of Hematology.

Entry and Trafficking of Granzyme B in Target Cells During Granzyme B-Perforin–Mediated Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1044-1054 ◽  
Author(s):  
Michael J. Pinkoski ◽  
Marita Hobman ◽  
Jeffrey A. Heibein ◽  
Kevin Tomaselli ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Indirect Immunofluorescence ◽  
Target Cell ◽  
Granzyme B ◽  
Fluorescein Isothiocyanate ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Direct Visualization ◽  
New Model ◽  
American Society

Abstract In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin–induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death. © 1998 by The American Society of Hematology.

scholarly journals Mouse and Human Granzyme B Have Distinct Tetrapeptide Specificities and Abilities to Recruit the Bid Pathway

2006 ◽  
Vol 282 (7) ◽  
pp. 4545-4552 ◽  
Author(s):  
Livia Casciola-Rosen ◽  
Margarita Garcia-Calvo ◽  
Herbert G. Bull ◽  
Joseph W. Becker ◽  
Tonie Hines ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Granzyme B ◽  
Target Cells ◽  
Cytotoxic Lymphocyte ◽  
Induce Cell Death ◽  
Important Mediator ◽  
Protease Substrate ◽  
Species Specific ◽  
And Function ◽  
Human And Mouse

Granzyme B is an important mediator of cytotoxic lymphocyte granule-induced death of target cells, accomplishing this through cleavage of Bid and cleavage and activation of caspases as well as direct cleavage of downstream substrates. Significant controversy exists regarding the primary pathways used by granzyme B to induce cell death, perhaps arising from the use of different protease/substrate combinations in different studies. The primary sequence of human, rat, and mouse granzymes B is well conserved, and the substrate specificity and crystal structure of the human and rat proteases are extremely similar. Although little is known about the substrate specificity of mouse granzyme B, recent studies suggest that it may differ significantly from the human protease. In these studies we show that the specificities of human and mouse granzymes B differ significantly. Human and mouse granzyme B cleave species-specific procaspase-3 more efficiently than the unmatched substrates. The distinct specificities of human and mouse granzyme B highlight a previously unappreciated requirement for Asp192 in the acquisition of catalytic activity upon cleavage of procaspase-3 at Asp175. Although human granzyme B efficiently cleaves human or mouse Bid, these substrates are highly resistant to cleavage by the mouse protease, strongly indicating that the Bid pathway is not a major primary mediator of the effects of mouse granzyme B. These studies provide important insights into the substrate specificity and function of the granzyme B pathway in different species and highlight that caution is essential when designing and interpreting experiments with different forms of granzyme B.

scholarly journals Human and murine granzyme B exhibit divergent substrate preferences

2007 ◽  
Vol 176 (4) ◽  
pp. 435-444 ◽  
Author(s):  
Sean P. Cullen ◽  
Colin Adrain ◽  
Alexander U. Lüthi ◽  
Patrick J. Duriez ◽  
Seamus J. Martin
Keyword(s):  
Granzyme B ◽  
Target Cells ◽  
Substrate Selection ◽  
Cytotoxic Lymphocyte ◽  
Substrate Preferences ◽  
Caspase Family ◽  
Direct Processing ◽  
Mitochondrial Permeabilization ◽  
Human And Mouse ◽  
Do So

The cytotoxic lymphocyte protease granzyme B (GzmB) can promote apoptosis through direct processing and activation of members of the caspase family. GzmB can also cleave the BH3-only protein, BID, to promote caspase-independent mitochondrial permeabilization. Although human and mouse forms of GzmB exhibit extensive homology, these proteases diverge at residues predicted to influence substrate binding. We show that human and mouse GzmB exhibit radical differences in their ability to cleave BID, as well as several other key substrates, such as ICAD and caspase-8. Moreover, pharmacological inhibition of caspases clonogenically rescued human and mouse target cells from apoptosis initiated by mouse GzmB, but failed to do so in response to human GzmB. These data demonstrate that human and murine GzmB are distinct enzymes with different substrate preferences. Our observations also illustrate how subtle differences in enzyme structure can radically affect substrate selection.

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

1981 ◽  
Vol 39 ◽  
pp. 452-453
Author(s):  
K. E. Muse ◽  
D. G. Fischer ◽  
H. S. Koren
Keyword(s):  
Target Cell ◽  
Human Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Limited Information ◽  
Blood Monocytes ◽  
Tumoricidal Activity ◽  
Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

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