This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

NOTE: Spleens were derived from mice (6-10 weeks of age) and all animal experiments were performed according to the animal ethics guidelines of the Peter MacCallum Cancer Centre (E486).
1. Preparation of Samples.
<ol>
	<li>Preparation of activated mouse NK cells.
	<ol>
		<li>Isolate primary naive NK cells from single-cell suspensions of the spleens of C57BL/6 or B6.GrzmB-/- (GzmB gene null) mice by negative selection using commercially available kits.</li>
		<li>Follow the manufacturers&#8217; protocol. Briefly, magnetically label &#8220;non-NK&#8221; cells such as T and B cells, dendritic cells, granulocytes, macrophages, and red blood cells using biotin-conjugated antibodies against their specific surface markers. Use magnetic separation with anti-Biotin beads to deplete &#8220;non-NK&#8221; cells.</li>
		<li>Culture isolated NK cells for 5-7 days in media containing IL-2 (1,000 U/ml) at 700,000 cells/ml in 24-well plates at 37&#160;&#176;C.</li>
		<li>Alternatively, use activated T cells 19, primary antigen-restricted CTL from OT1 T cell receptor transgenic mice 20, CTL generated in mixed lymphocyte cultures 21, or supernatants from activated NK cells to determine the activity of secreted GzmB 19.</li>
	</ol>
	</li>
	<li>Maintenance of human NK cell line and isolation of primary NK cells.
	<ol>
		<li>Maintain human NK leukemia (YT cells) in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum. Alternatively, purify primary NK cells from the peripheral blood of healthy subjects and stimulate for 2-3 days in medium containing IL-2 at 100 U/ml as previously described 22.</li>
	</ol>
	</li>
	<li>Generation of cell lysates.
	<ol>
		<li>Wash cells three times in PBS (pH 7.4), then suspend in Nonidet P-40 lysis buffer (0.1% NP-40, 250 mM NaCl, 25 mM Hepes, 2.5 mM EDTA). Ideally, lyse a minimum of 5 x 106 NK cells, or 1 x 107 T cells in 100 &#181;l buffer. Do not add protease inhibitors, as many are irreversible inhibitors of serine proteases, and in particular tryptases such as GzmA. Incubate on ice for 20 min, and then pellet the nuclei in a microcentrifuge at 15,000 x g for 10 min. Discard the nuclear pellet and transfer the supernatant to a fresh tube.</li>
	</ol>
	</li>
	<li>Determine the protein concentration of the lysates.
	<ol>
		<li>Use a commercially available protocol of choice such as, Bradford or bicinchoninic acid (BCA) and determine the protein concentration. Ensure that the concentration is ~2 mg/ml minimum. Store lysates at -20 &#176;C until required (granzyme activity is stable for many months at -20 &#176;C).</li>
	</ol>
	</li>
</ol>
2. Granzyme B Activity Assay
<ol>
	<li>Preparation of Reagents
	<ol>
		<li>Prepare a 10 mM stock of the substrates (Boc-AAD-S-Bzl or Ac-IEPD-pNA) in DMSO (~5 mg/ml) and store in aliquots at -20 &#176;C. Prepare a working dilution freshly and discard after each day, as Boc-AAD-S-Bzl partially hydrolyzes on storage in DMSO.</li>
		<li>Prepare a 250 mM stock of the colorimetric reagent (5,5&#8217;-dithio-bis(2-nitrobenzoic acid), DTNB) in DMSO (0.09 g/ml) and store at -20 &#176;C. Prepare a fresh working dilution each day. This reagent is only required for substrates with SBzl indicator groups, not pNA.</li>
		<li>Make up fresh buffer (0.1M HEPES, 0.05 M MgCl2, pH 7.3) every 2-3 weeks.</li>
	</ol>
	</li>
	<li>Bring DTNB and synthetic substrates to room temperature. Dilute substrates (1:16, e.g. 100 &#181;l in 1.6 ml of buffer) and DTNB (1:250, e.g. 10 &#181;l in 2.5 ml buffer) and mix well.</li>
	<li>Dilute a minimum of 100 &#181;g protein lysate of each sample in buffer to a final volume of 160 &#181;l. Using a multichannel pipette add 50 &#181;l per well in triplicate (~33.3 &#181;g/well) in a &#8216;&#8216;U&#8217;&#8217; bottom vinyl 96-well plate. Include a &#8220;buffer-only&#8221; control (also in triplicate).</li>
	<li>Add 100 &#181;l of diluted DTNB to the samples/buffer control (omit this step when working with Ac-IEPD-pNA. 
	NOTE: Cleavage of Ac-IEPD-pNA by GzmB directly releases fluorogenic pNA (Figure 4B).</li>
	<li>Use a multichannel pipette to quickly add 50 &#181;l of diluted substrates to each set of triplicates, starting with the buffer then any negative controls and finishing with expected positive samples.</li>
	<li>Place the plate in the plate reader. Measure Asp-ase activity by hydrolysis of Boc-Ala-Ala-Asp-S-Bzl/Ac-IEPD-pNA in a microplate reader at OD 405 nm. Use a kinetic assay protocol, take 25 readings, every 15 sec (ca. 6 min total reading time).</li>
	<li>Use the absorbance readings generated at each time point to manually calculate the rate of substrate cleavage if the plate-reader&#8217;s software cannot generate a value for Vmax.</li>
</ol>
3. Analysis of Data
NOTE: The data generated is presented as maximum velocity, defined as change of OD over time (mOD/min).
<ol>
	<li>To analyze the data, determine the maximum rate of substrate cleavage, which is calculated from the rate of change in absorbance. Do this automatically using the plate reader software, which typically has this option.</li>
	<li>Alternatively after viewing the kinetic plots of change in absorbance, which are generated during the kinetic assay on the plate reader, manually select the data points which give the steepest straight line. For most assays the maximum rate is achieved within the first 2 min of the assay.</li>
	<li>Subtract the initial OD reading from the highest OD reading on this straight line and divide by the time interval. Transfer raw data to Microsoft Excel/Graph pad for statistical analysis and graphic display.</li>
</ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+lymphocyte+granules+are+secretory+lysosomes,+containing+both+perforin+and+granzymes.">Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.</a> J Exp Med. 173 (5), 1099-1109 (1991).</li><li>Berthou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granzyme+B+and+Fas+ligand+proteins+by+human+keratinocytes+contributes+to+epidermal+cell+defense.">Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense.</a> J Immunol. 159 (11), 5293-5300 (1997).</li><li>Tschopp, C. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mast+cells+produce+and+release+the+cytotoxic+lymphocyte+associated+protease+granzyme+B+upon+activation.">Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.</a> Mol Immunol. 44 (14), 3462-3472 (2007).</li><li>Jahrsdorfer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B+produced+by+human+plasmacytoid+dendritic+cells+suppresses+T+cell+expansion.">Granzyme B produced by human plasmacytoid dendritic cells suppresses T cell expansion.</a> Blood. , (2009).</li><li>Hagn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+cells+secrete+granzyme+B+when+recognizing+viral+antigens+in+the+context+of+the+acute+phase+cytokine+IL-21.">Human B cells secrete granzyme B when recognizing viral antigens in the context of the acute phase cytokine IL-21.</a> J Immunol. 183 (3), 1838-1845 (2009).</li><li>Hagn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+cells+differentiate+into+granzyme+B-secreting+cytotoxic+B+lymphocytes+upon+incomplete+T-cell+help.">Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help.</a> Immunol Cell Biol. 90 (4), 457-467 (2012).</li><li>Froelich, C. 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The authors have nothing to disclose.

Historically the granzymes were identified as key effector molecules of cytotoxic lymphocytes (CTL and NK cells) capable of inducing a rapid apoptotic death in target cells. This was principally due to the action of GzmB, which cleaved target substrate molecules at aspartate (D) residues and thus was able to activate the caspase cascade by both cleaving pro-caspases, as well as several of their downstream targets. However, it is now appreciated that GzmB expression is not confined to lymphoid cytotoxic cells and its func...

Granzymes are a family of serine proteases found in the secretory lysosomes of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) 1. Five different granzymes exist in humans (A, B, H, K and M), and ten in mice (A - G, K, M and N) 2,3. Granzyme A and Granzyme B (GzmA, GzmB) are the most abundant and have been extensively investigated in the human and rodent setting.
The classic function of GzmB is the induction of apoptosis in target cells executed in conjunction with the pore-forming protein perforin, which permits the granzyme to access the target cell cytosol 4. Although GzmB expression is unequivocally found in cytotoxic lymphocytes, recent studies have been addressing a variety of other GzmB-expressing cell types, including but not limited to keratinocytes 5, basophils 6, mast cells 7, plasmacytoid dendritic cells 8, and B cells 9,10. In this context, non-apoptotic GzmB functions were revealed ranging from participation in inflammatory processes, tissue remodelling and other immunoregulatory properties 11-14.
Given that a broader biological role has been proposed for GzmB than previously suspected, researchers require reliable and specific tools for its detection. Of advantage is GzmB&#8217;s specific requirement to cleave on the carboxyl side of aspartic acid residues, a property unique among eukaryotic serine proteases 15. Mouse, human and rat GzmB are structurally very similar, however the extended substrate specificity of mouse GzmB differs subtly from that of both human and rat 16, which means that certain generic substrates with Asp at the terminal (P1) can be cleaved efficiently by GzmB from all three species, whereas other substrates with more complicated sequences upstream of P1 may give widely variable results. In both the past and more recent literature, this fact has caused considerable confusion and misinterpretation of the biological significance of some experimental findings, even though carefully controlled, kinetic studies have sought to correct the situation 17.
In this paper we have sought to illustrate these points using two commercially available substrates, namely Boc-Ala-Ala-Asp-SBzl and N-acetyl-Ile-Glu-Pro-Asp-p-nitroanilide. The two reagents do generate different reactive groups following cleavage (a free sulphydryl versus a fluorescent free paranitroanilide), but this has no effect whatever on proteolytic cleavage. The described protocol is a modern adaption of a very old protocol 18, but should help investigators to use the different GzmB substrates appropriately, while also providing a methodological framework for detecting the activity of other granzymes, such as GzmA and GzmH.

<table border="0" cellpadding="0" cellspacing="0" width="1133">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Product</td>
			<td style="width:241px;">Company</td>
			<td style="width:181px;">Catalogue number</td>
			<td style="width:427px;">Comment/description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boc-Ala-Ala-Asp-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMSB05</td>
			<td>Granzyme B substrate (mouse and human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ac-IEPD-pNA&#160;</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMPNA009</td>
			<td>Granzyme B substrate (only human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-a-CBZ-L-lysine-S-Bzl</td>
			<td>Sigma-Aldrich</td>
			<td>C3647</td>
			<td>Granzyme A substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suc-Phe-Leu-Phe-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SB025</td>
			<td>Granzyme H substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5,5&#8217;-dithio-bis(2-nitrobenzoic acid)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td>&#160;DTNB, Ellman&#8217;s Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit II mouse</td>
			<td>Miltenyi Biotec GmbH</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-657</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td>Biorad iMark</td>
			<td></td>
			<td>Biorad Microplate Manager Software Version MPM6.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serocluster U-bottom vinyl 96-well plate</td>
			<td>Corning, MA, USA</td>
			<td>2797</td>
			<td></td>
		</tr>
	</tbody>
</table>

a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

The Boc-AAD-S-Bzl substrate is specific for GzmB
The serine protease GzmB is a major constituent of cytotoxic lymphocytes (CTL and NK cells) and is predominantly responsible for inducing rapid apoptotic death in target cells, such as virus-infected or transformed cells. This is largely due to its substrate preference for cleavage after certain specific aspartate residues in selected proteins, an attribute shared with the caspases, which also cleave after aspartate residues but...

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A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

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Research

JoVE Journal

Chemistry

12.9K Views.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Video: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Cancer Research

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

Biochemistry

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Immunology and Infection

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Biology

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://app.jove.com/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Blot Techniques

Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels

Source: Deshmukh, A. A. et al. <a href="https://www.jove.com/video/58938" target="_blank">Detection of Protease Activity by Fluorescent Peptide Zymography.</a> J. Vis. Exp. (2019)
In this video, we perform zymography, an electrophoretic technique, to analyze the proteolytic activity within biological samples.

Source: Deshmukh, A. A. et al. Detection of Protease Activity by Fluorescent Peptide Zymography. J. Vis. Exp. (2019)
In this video, we perform zymography, ...

Cancers of the Nervous System

In vitro studies

A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

This video demonstrates an assay to monitor neutrophil activation and granule exocytosis through a degranulation assay. Upon activation by proinflammatory compounds, cytoplasmic granules within neutrophils containing gelatinase fuse with the cell membrane, discharging their cargo. The presence of gelatinase in the supernatant is assessed through the hydrolysis of a fluorophore-conjugated gelatin substrate, confirming neutrophil activation and degranulation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare mouse bone marrow ...

Immunology

Immunodiagnostics

Specialized Assays

An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation

This video demonstrates a degranulation assay of azurophilic granules in mouse neutrophils, involving the stimulation of neutrophils to release myeloperoxidase-containing azurophilic granules. The subsequent enzymatic reaction is employed to quantify myeloperoxidase levels, providing insight into neutrophil activation.