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.

참고 : 마우스에서 파생 된 비장 (나이 6-10 주) 모든 동물 실험은 피터 맥 칼럼 암 센터 (E486)의 동물 윤리 지침에 따라 수행 하였다. 샘플 1. 준비. <ol><li> 활성화 마우스 NK 세포의 준비. <ol><li> 시판되는 키트를 사용하여 음성 선별에 의해 (GzmB 유전자 NULL) 마우스 - / - C57BL / 6 또는 B6.GrzmB의 비장의 단일 세포 현탁액으로부터 기본 나이브 NK 세포를 분리. </li><li> 제조 업체의 프로토콜을 따르십시오. 요약하면, 이러한 자기 T 및 B 세포, 수지상 세포, 과립구, 대 식세포, 및 특정 표면 마커에 대해 바이오틴 - 컨쥬 게이트 항체를 사용하여 적혈구 세포와 같은 &quot;비 - NK&quot;세포를 라벨. &quot;비 NK&quot;세포를 고갈 방지 비오틴 구슬 자기 분리를 사용합니다. </li><li> 배양은 24 웰 플레이트에서 700,000 세포 / ml에서 IL-2 (1000 U / ㎖)를 함유하는 미디어 5-7 일간 NK 세포를 격리37 &#160; ° C. </li><li> 대안 적 사용은 19 T 세포, OT1 T 세포 수용체 트랜스 제닉 마우스 (20)로부터 기본 항원 제한된 CTL이 CTL 분비 GzmB (19)의 행동을 결정하기 위하여 활성화 된 NK 세포로부터 혼합 림프구 배양 물 (21), 또는 상층 액에 생성 된 활성화. </li></ol></li><li> 인간 NK 세포주 및 일차 NK 세포의 분리의 유지. <ol><li> 10 % 우태 혈청으로 보충 된 로스웰 파크 메모리얼 연구소 (RPMI) 배지에서 인간 NK 백혈병 (YT 세포)를 유지한다. 또한, 정상인의 말초 혈액에서 차 NK 세포를 정화하고 이전 22 설명 된 바와 같이 IL-2 100 U / ml의에서이 함유 배지 2 ~ 3 일 동안 자극한다. </li></ol></li><li> 세포 용 해물의 생성. <ol><li> PBS (PH 7.4)에 세포를 세 번 세척 한 후 Nonidet P-40 용해 버퍼에 일시 중단 (0.1 % NP-40, 250 mM의 염화나트륨, 25 mM의 헤 페스, 2.5 mM의 EDTA). 이상적으로는, 5 × 106의 최소 용해 </sNK 세포, 또는 100 μL 버퍼에 1 × 10 7 T 세포&gt;까지. 프로테아제 억제제를 추가하지 마십시오, 많은는 GzmA 취소 할 수없는 세린 프로테아제 억제제, 특히 tryptases에 있습니다. 얼음에서 20 분 동안 인큐베이션 한 후 10 분간 15,000 XG에 미세 원심에서 핵 펠렛. 핵 펠렛을 취소하고 새로운 튜브에 뜨는을 전송합니다. </li></ol></li><li> 해물의 단백질 농도를 결정합니다. <ol><li> 이러한 브래드 bicinchoninic 또는 산 (BCA)과 같은 선택의 시판 프로토콜을 사용하여 단백질 농도를 결정한다. 농도가 1 ~ 2 ㎎ / ㎖ 최소 있는지 확인하십시오. 필요한 때까지 -20 ° C에서 보관 용 해물 (그랜 자임 활동은 -20 ° C에서 몇 달 동안 안정). </li></ol></li></ol> 2. 그랜 자임 B 활동 분석 <ol><li> 시약의 조제 <ol><li> DMSO에 기판 (BOC-AAD-S-BZL 또는 AC-IEPD-pNA를)의 10 mM의 주식을 준비합니다 (~ 5 ㎎ / ㎖) -20 &amp; #에서 분취에 저장176; C. 갓 작업 희석을 준비하고 BOC-AAD-S-BZL 부분적으로 DMSO에 저장에 가수 분해로, 하루 후 폐기합니다. </li><li> -20 ° C에서 250 밀리미터의 DMSO 비색 시약 (5,5&#39;- 디티 오 - 비스 (2- 니트로 벤조산), DTNB)의 재고 (0.09 g / mL) 및 저장소를 준비한다. 매일 신선한 작업 희석을 준비합니다. 이 시약은 SBzl 표시가 아닌 그룹 pNA를 가진 기판이 필요합니다. </li><li> 신선한 버퍼 (0.1M HEPES, 0.05 M의 MgCl 2, pH를 7.3) 2 ~ 3 주까지합니다. </li></ol></li><li> 실온으로 DTNB 및 합성 기판을 준비한다. 희석 기판 (1시 16분, 예를 들어 1.6 버퍼 용액에 100 μL)과 DTNB (1 : 250, 예를 들어 10 2.5 ml의 버퍼의 μL) 잘 섞는다. </li><li> 160 μL의 최종 부피 버퍼에 각 시료 100 μg의 단백질 용 해물의 최소 희석. 멀티 채널 피펫을 사용하여 &#39;&#39;U &#39;&#39;바닥 비닐 96 웰 플레이트 (~ 33.3 ㎍ / 웰) 세중 잘 당 50 μl를 추가합니다.&quot;버퍼 전용&quot;제어를 포함 (또한 세중). </li><li> AC-IEPD-pNA를 사용하여 작업 할 때 샘플 / 버퍼 제어로 희석 DTNB 100 μl를 추가합니다 (이 단계를 생략합니다. 참고 : GzmB에 의해 AC-IEPD-pNA를의 쪼개짐 직접 형광 pNA를 (그림 4B)를 발표한다. </li><li> 빨리 다음 버퍼 부정적인 컨트롤로 시작하는 예상 양의 샘플을 마무리, 삼중의 각 세트로 희석 기판의 50 μl를 추가하는 멀티 채널 피펫을 사용합니다. </li><li> 플레이트 리더에서 접시를 놓습니다. OD 405 nm에서 마이크로 플레이트 리더에 BOC-알라 - 알라 -의 ASP-S-BZL / AC-IEPD-pNA를 가수 분해하여 ASP를-ASE 활동을 측정한다. 25 판독, 매 15 초 (약 6 분 총 독서 시간)을 가지고, 운동 분석 프로토콜을 사용합니다. </li><li> 플레이트 리더 (VMAX)의 소프트웨어에 대한 값을 생성 할 수없는 경우 직접 기판의 절단 속도를 계산하기 위해 각 시점에서 생성 흡광도 판독 값을 사용한다. </li></ol> 다 3. 분석타 참고 : 시간이 지남에 OD의 변화 (MOD / 분)로 정의 된 최대 속도로 제시됩니다 생성 된 데이터,. <ol><li> 데이터를 분석하는, 흡광도의 변화율로부터 산출되는 기판 절단의 최대 속도를 결정한다. 자동 일반적으로이 옵션이 플레이트 리더 소프트웨어를 사용하여이 작업을 수행합니다. </li><li> 또는 수동으로 플레이트 리더에 운동 분석 중에 생성되는 흡광도의 변화의 운동 플롯을 본 후 가파른 직선을주는 데이터 포인트를 선택합니다. 대부분의 상기 분석 최대 레이트로는 분석의 제 2 분 이내에 달성된다. </li><li> 시간 간격으로이 직선과 분열의 가장 높은 OD 독서에서 초기 OD 읽기를 뺍니다. 통계 분석 및 그래픽 디스플레이 용 Microsoft Excel / 그래프 패드에 원시 데이터를 전송합니다. </li></ol>

<ol>
	<li>Trapani, J. A., Browne, K. A., Dawson, M., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+functional+Asp-ase+(natural+killer+cell+granzyme+B)+using+a+monoclonal+antibody).">Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody).</a> Biochem Biophys Res Commun. 195 (2), 910-920 (1993).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quarter+century+of+granzymes.">A quarter century of granzymes.</a> Cell Death Differ. 19 (1), 28-35 (2012).</li><li>Hoves, S., Trapani, J. A., Voskoboinik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+battlefield+of+perforin/granzyme+cell+death+pathways.">The battlefield of perforin/granzyme cell death pathways.</a> J Leukoc Biol. , (2009).</li><li>Peters, P. 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. 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=Granzyme+B,+a+novel+mediator+of+allergic+inflammation:+its+induction+and+release+in+blood+basophils+and+human+asthma.">Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma.</a> Blood. 108 (7), 2290-2299 (2006).</li><li>Strik, M. 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. J., Pardo, J., Simon, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule-associated+serine+proteases:+granzymes+might+not+just+be+killer+proteases.">Granule-associated serine proteases: granzymes might not just be killer proteases.</a> Trends Immunol. 30 (3), 117-123 (2009).</li><li>Hagn, M., Jahrsdorfer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+human+B+cells+secrete+granzyme+B?+Insights+into+a+novel+B-cell+differentiation+pathway.">Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.</a> Oncoimmunology. 1 (8), 1368-1375 (2012).</li><li>Susanto, O., Trapani, J. A., Brasacchio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+granzyme+biology.">Controversies in granzyme biology.</a> Tissue Antigens. 80 (6), 477-487 (2012).</li><li>Walch, 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=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157 (6), 1309-1323 (2014).</li><li>Odake, S., 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+and+murine+cytotoxic+T+lymphocyte+serine+proteases:+subsite+mapping+with+peptide+thioester+substrates+and+inhibition+of+enzyme+activity+and+cytolysis+by+isocoumarins.">Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.</a> Biochemistry. 30 (8), 2217-2227 (1991).</li><li>Casciola-Rosen, L., 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=Mouse+and+human+granzyme+B+have+distinct+tetrapeptide+specificities+and+abilities+to+recruit+the+bid+pathway.">Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway.</a> J Biol Chem. 282 (7), 4545-4552 (2007).</li><li>Kaiserman, D., 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=The+major+human+and+mouse+granzymes+are+structurally+and+functionally+divergent.">The major human and mouse granzymes are structurally and functionally divergent.</a> J Cell Biol. 175 (4), 619-630 (2006).</li><li>Powers, J. C., Kam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+thioester+substrates+for+serine+peptidases+and+metalloendopeptidases.">Peptide thioester substrates for serine peptidases and metalloendopeptidases.</a> Methods Enzymol. 248, 3-18 (1995).</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=Activated+mouse+B+cells+lack+expression+of+granzyme.">Activated mouse B cells lack expression of granzyme.</a> B. J Immunol. 188 (2), 3886-3892 (2012).</li><li>Konjar, S., 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+and+mouse+perforin+are+processed+in+part+through+cleavage+by+the+lysosomal+cysteine+proteinase+cathepsin+L.">Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L.</a> Immunology. 131 (2), 257-267 (2010).</li><li>Sutton, V. R., 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=Residual+active+granzyme+B+in+cathepsin+C-null+lymphocytes+is+sufficient+for+perforin-dependent+target+cell+apoptosis.">Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis.</a> J Cell Biol. 176 (4), 425-433 (2007).</li><li>Trapani, J. A., Smyth, M. J., Apostolidis, V. A., Dawson, M., Browne, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule+serine+proteases+are+normal+nuclear+constituents+of+natural+killer+cells.">Granule serine proteases are normal nuclear constituents of natural killer cells.</a> J Biol Chem. 269 (28), 18359-18365 (1994).</li><li>Cullen, S. P., Adrain, C., Luthi, A. U., Duriez, P. J., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+granzyme+B+exhibit+divergent+substrate+preferences.">Human and murine granzyme B exhibit divergent substrate preferences.</a> J Cell Biol. 176 (4), 435-444 (2007).</li><li>Thornberry, N. A., 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=A+combinatorial+approach+defines+specificities+of+members+of+the+caspase+family+and+granzyme+B.+Functional+relationships+established+for+key+mediators+of+apoptosis.">A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.</a> J Biol Chem. 272 (29), 17907-17911 (1997).</li><li>Bird, C. H., 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=Selective+regulation+of+apoptosis:+the+cytotoxic+lymphocyte+serpin+proteinase+inhibitor+9+protects+against+granzyme+B-mediated+apoptosis+without+perturbing+the+Fas+cell+death+pathway.">Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.</a> Mol Cell Biol. 18 (11), 6387-6398 (1998).</li><li>Bots, M., Medema, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serpins+in+T+cell+immunity.">Serpins in T cell immunity.</a> J Leukoc Biol. 84 (5), 1238-1247 (2008).</li><li>Sun, 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=A+new+family+of+10+murine+ovalbumin+serpins+includes+two+homologs+of+proteinase+inhibitor+8+and+two+homologs+of+the+granzyme+B+inhibitor+(proteinase+inhibitor+9).">A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9).</a> J Biol Chem. 272 (24), 15434-15441 (1997).</li><li>Bird, C. H., Hitchen, C., Prescott, M., Harper, I., Bird, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodetection+of+granzyme+B+tissue+distribution+and+cellular+localisation.">Immunodetection of granzyme B tissue distribution and cellular localisation.</a> Methods Mol Biol. 844, 237-250 (2012).</li><li>Jenkins, M. R., 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=Visualizing+CTL+activity+for+different+CD8++effector+T+cells+supports+the+idea+that+lower+TCR/epitope+avidity+may+be+advantageous+for+target+cell+killing.">Visualizing CTL activity for different CD8+ effector T cells supports the idea that lower TCR/epitope avidity may be advantageous for target cell killing.</a> Cell Death Differ. 16 (4), 537-542 (2009).</li><li>Edwards, K. M., Kam, C. M., Powers, J. C., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+cytotoxic+T+cell+granule+serine+protease+granzyme+H+has+chymotrypsin-like+(chymase)+activity+and+is+taken+up+into+cytoplasmic+vesicles+reminiscent+of+granzyme+B-containing+endosomes.">The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.</a> J Biol Chem. 274 (43), 30468-30473 (1999).</li><li>Sutton, V. R., 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=Initiation+of+apoptosis+by+granzyme+B+requires+direct+cleavage+of+bid,+but+not+direct+granzyme+B-mediated+caspase+activation.">Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.</a> J Exp Med. 192 (10), 1403-1414 (2000).</li></ol>

The authors have nothing to disclose.

역사적으로 granzymes은 표적 세포의 급속한 세포 사멸 죽음을 유도 할 수있는 세포 독성 림프구 (CTL 및 NK 세포)의 주요 이펙터 분자로 확인되었다. 이는 아스 파르 테이트 (D) 잔기에 타깃 기판 분자를 절단함으로써 그 하류 표적 여러뿐만 아니라, 모두 절단 프로 카스파하여 카스파 제 캐스케이드를 활성화 할 수 있었다 GzmB의 작용에 주로이었다. 그러나, 지금 GzmB 발현 림프구 세포 독성 세포에 국한...

Granzymes 자연 킬러 (NK) 세포 및 세포 독성 T 림프구 (CTL) (1)의 분비 리소좀에서 발견되는 세린 프로테아제 패밀리이다. 다섯 가지 granzymes 인간 (A, B, H, K 및 M)에 존재하고, 마우스에서 십 (A - G, K, M 및 N) 2,3-. 그랜 자임과 그랜 자임 B (GzmA, GzmB)는 가장 풍부하고 광범위하게 인간과 설치류 설정에서 조사되었다. GzmB의 고전 함수는 표적 세포 사이토 졸로 액세스 4 그랜 자임을 허용 기공 형성 단백질 퍼포과 함께 실행되는 표적 세포에서 아폽토시스의 유도이다. GzmB 발현 명백하게 세포 상해 성 림프구에서 발견되지만, 최근의 연구는 호염기구 (6), 비만 세포 (7) 형질 수지상 세포 (8) 및 B 세포 (9)를 포함하지만, 케 라티노 사이트 (5)에 한정되지 않고, 다른 GzmB 발현 세포 유형의 다양한 어드레싱되었습니다 10.이러한 맥락에서, 비 세포 사멸 GzmB 기능은 염증 과정, 조직 리모델링과 다른 면역 특성 11-14 참여에 이르기까지 드러났다. 폭 넓은 생물학적 역할이 이전에 의심보다 GzmB 제안 된 것을 감안할 때, 연구자는 검출 신뢰성과 특정 도구가 필요합니다. 이점 진핵 세린 프로테아제 (15) 사이에서 고유 아스파르트 산 잔기, 속성의 카복실 측 쪼개지 GzmB의 특정 요구 사항이다. 마우스는 인간과 쥐 GzmB 그러나 마우스 GzmB의 확장 기질 특이성 터미널 (P1)에서의 ASP와 특정 일반 기판 GzmB 효율적으로 절단 할 수 있음을 의미 모두 인간과 쥐 16과는 미묘하게 다른, 구조적으로 매우 유사 세 종, 상류 P1의 더 복잡한 시퀀스와 다른 기판 반면 널리 변수 결과를 제공 할 수 있습니다. 과거 최신 리튬 둘terature,이 사실은주의 깊게 통제에도 불구하고, 상당한 혼란과 일부 실험 결과의 생물학적 의미의 오해가 발생했습니다, 운동 연구는 상황 (17)를 해결하기 위해 노력했다. 이 논문에서 우리는 두 가지 상업적으로 이용 가능한 기판, 즉 BOC-알라 - 알라 -의 ASP SBzl 및 N- 아세틸 일드 서열 1 프로의 ASP-P-니트로 아닐리드를 사용하여 이러한 점을 설명하기 위해 노력했다. 이 시약은 분열 (형광 무료 paranitroanilide 대 무료 sulphydryl) 다음과 같은 서로 다른 반응 그룹을 생성 할 수 있지만,이 단백질 분해 분열에 영향 무엇이든이 없습니다. 기술 된 프로토콜은 매우 오래 프로토콜 (18)의 현대 적응이지만 또한 GzmA 및 GzmH 다른 granzymes의 활성을 검출하기위한 방법 론적 틀을 제공하면서 조사관, 적절히 다른 GzmB 기판을 사용하는 것이 도움이 될 것이다.

<table><tbody><tr><td>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><td>Ac-IEPD-pNA&amp;nbsp;</td><td>SM Biochemicals LLC, CA</td><td>SMPNA009</td><td>Granzyme B substrate (only human)</td></tr><tr><td>N-&amp;#945;-CBZ-L-lysine-S-Bzl</td><td>Sigma-Aldrich</td><td>C3647</td><td>Granzyme A substrate</td></tr><tr><td>Suc-Phe-Leu-Phe-S-Bzl</td><td>SM Biochemicals LLC, CA</td><td>SB025</td><td>Granzyme H substrate</td></tr><tr><td>5,5&amp;rsquo;-dithio-bis(2-nitrobenzoic acid)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8130</td><td>DTNB, Ellman&amp;rsquo;s Reagent</td></tr><tr><td>NK cell isolation kit II mouse</td><td>Miltenyi Biotec GmbH</td><td>130-096-892</td><td>negative selection kit</td></tr><tr><td>NK cell isolation kit human</td><td>Miltenyi Biotec GmbH</td><td>130-092-657</td><td>negative selection kit</td></tr><tr><td>Plate reader</td><td>Biorad iMark</td><td></td><td>Biorad Microplate Manager Software Version MPM6.3</td></tr><tr><td>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.

BOC-AAD-S-BZL 기판 GzmB에 대한 특정 세린 프로테아제 GzmB 세포 독성 림프구 (CTL 및 NK 세포)의 주요 구성 성분이며, 바이러스 감염 또는 형질 전환 세포와 같은 표적 세포에 빠른 세포 사멸 죽음을 유도 주로 담당하고 있습니다. 이 때문에 선택된 단백질의 특정 특정 아스파 테이트 잔류 후 분열에 대한 기판 선호도에 크게이며, 속성은 아스 파르 테이트 잔류 후 절단?...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

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.

BOC-알라 - 알라 -의 ASP-S-BZL의 가수 분해 : 색채 특히 그랜 자임 B 단백질 분해 활동를 측정 분석

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

a-colorimetric-assay-that-specifically-measures-granzyme-b

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

13.0K 조회수.  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.

비디오: 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.

형광 성 펩 티 드 Zymography에 의해 프로 테아 제 활동의 탐지

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

연구

암 연구학

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.

이 단지에서 CD95 사망 유도 신호 복합체 및 프로카스파스-8의 처리 구성 측정

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

생화학

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.

정성 및 정량 검출을위한 시험 법 및 단백질 항생제 특성

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

면역학 및 감염병학

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.

형광 분석 또는 유세포분석을 사용한 카스파제 활성 측정

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

Biophysical Assay를 사용한 PROTAC의 삼원 복합체 형성 특성 분석

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.

저자 스포트라이트: PROTACS 삼원 복합체 특성화를 위한 생물물리학 분석 평가 

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).

단백질 분해 표적 키메라(PROTACS)에 의해 유도된 삼원 복합체 형성을 평가하기 위한 생물물리학 분석법의 개발 및 적용

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.

포유 동물 세포에서 인간의 Granzymes의 높은 수율과 비용 효율적인 발현 시스템

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

생물학

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://appjove.unicartagenaproxy.elogim.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.

단백질 가공을 위한 화학발광 웨스턴 블롯 분석

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

A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Source: Jaimes, J. A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

BOC-알라 - 알라 -의 ASP-S-BZL의 가수 분해 : 색채 특히 그랜 자임 B 단백질 분해 활동를 측정 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

형광 성 펩 티 드 Zymography에 의해 프로 테아 제 활동의 탐지

이 단지에서 CD95 사망 유도 신호 복합체 및 프로카스파스-8의 처리 구성 측정

정성 및 정량 검출을위한 시험 법 및 단백질 항생제 특성

형광 분석 또는 유세포분석을 사용한 카스파제 활성 측정

단백질 분해 표적 키메라(PROTACS)에 의해 유도된 삼원 복합체 형성을 평가하기 위한 생물물리학 분석법의 개발 및 적용

단백질 분해 표적 키메라(PROTACS)에 의해 유도된 삼원 복합체 형성을 평가하기 위한 생물물리학 분석법의 개발 및 적용

단백질 분해 표적 키메라(PROTACS)에 의해 유도된 삼원 복합체 형성을 평가하기 위한 생물물리학 분석법의 개발 및 적용

포유 동물 세포에서 인간의 Granzymes의 높은 수율과 비용 효율적인 발현 시스템

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프로테아제의 단백질 분해 활성을 스크리닝하기 위한 형광 펩타이드 절단 분석