Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System

Xia, Zhinan; Kam, Chih-Min; Huang, Chao‐Song; Powers, James C.; Mandle, Robert J.; Stevens, Richard L; Lieberman, Judy

doi:10.1006/bbrc.1998.8102

Cited by 40 publications

(14 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recombinant GzmA, inactive SAGzmA, and GzmB were produced and purified as previously reported (8,65). PFP was purified from the rat RNK-16 cell line and used at a sublytic concentration, titrated to induce Ͻ10% cytolysis, as described previously (44).…”

Section: Methodsmentioning

confidence: 99%

HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

Fan

Beresford

Zhang

et al. 2002

Molecular and Cellular Biology

Self Cite

124

View full text Add to dashboard Cite

The cytotoxic T-lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-stranded nicking is observed instead of oligonucleosomal fragmentation. A 270-to 420-kDa endoplasmic reticulum-associated complex (SET complex) containing the nucleosome assembly protein SET, the tumor suppressor pp32, and the base excision repair enzyme APE can induce single-stranded DNA damage in isolated nuclei in a granzyme A-dependent manner. The normal functions of the SET complex are unknown, but the functions of its components suggest that it is involved in activating transcription and DNA repair. We now find that the SET complex contains DNA binding and bending activities mediated by the chromatin-associated protein HMG2. HMG2 facilitates assembly of nucleoprotein higher-order structures by bending and looping DNA or by stabilizing underwound DNA. HMG2 is in the SET complex and coprecipitates with SET. By confocal microscopy, it is observed that cytoplasmic HMG2 colocalizes with SET in association with the endoplasmic reticulum, but most nuclear HMG2 is unassociated with SET. This physical association suggests that HMG2 may facilitate the nucleosome assembly, transcriptional activation, and DNA repair functions of SET and/or APE. HMG2, like SET and APE, is a physiologically relevant granzyme A substrate in targeted cells. HMG1, however, is not a substrate. Granzyme A cleavage after Lys65 in the midst of HMG box A destroys HMG2-mediated DNA binding and bending functions. Granzyme A cleavage and functional disruption of key nuclear substrates, including HMG2, SET, APE, lamins, and histones, are likely to cripple the cellular repair response to promote cell death in this novel caspase-independent death pathway.

show abstract

Section: Methodsmentioning

confidence: 99%

HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

Fan

Beresford

Zhang

et al. 2002

Molecular and Cellular Biology

Self Cite

124

View full text Add to dashboard Cite

show abstract

“…Recombinant GzmA, inactive S-AGzmA, and GzmB were produced and PFP was purified from RNK-16 cells as described (8,15,36,37). For loading, PFP was added at a sublytic concentration, determined for each cell as that needed to induce Յ10% lysis in 2 h (8).…”

Section: Methodsmentioning

confidence: 99%

Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis

Zhang

Beresford

Greenberg

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

150

View full text Add to dashboard Cite

Cytotoxic T lymphocytes (CTL) induce apoptosis by engaging death receptors or by exocytosis of cytolytic granules containing granzyme (Gzm) proteases and perforin. The lamins, which maintain the structural integrity of the nuclear envelope, are cleaved by caspases during caspase-mediated apoptosis. Although death receptor engagement and GzmB activate caspases, CTL also induce apoptosis during caspase blockade. Both GzmA and GzmB directly and efficiently cleave laminB in vitro, in situ in isolated nuclei and in cells loaded with perforin and Gzms, even in the presence of caspase inhibitors. LaminB is cleaved by GzmA at concentrations of 3 nM, but GzmB is 50 times less active. GzmA cuts laminB at R392; GzmB cuts at the caspase VEVD231 site. Characteristic laminB fragments generated by Gzm proteolysis also are observed during CTL lysis, even in the presence of caspase inhibitors or in cells overexpressing bcl-2. Lamins A͞C are direct substrates of GzmA, but not GzmB. GzmA and GzmB therefore directly target critical caspase substrates in caspase-resistant cells.

show abstract

“…14,33 All experiments using cells from knockout mice were performed with the approval of the Animal Care and Use Committee of the Immune Disease Institute and Harvard Medical School. Human PARP-1 was expressed in Escherichia coli as a His 6 tag fusion protein from pET5a-PARP-1, a gift from P. Kannan (Case University), and purified, as described.…”

Section: Recombinant and Purified Proteinsmentioning

confidence: 99%

The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5′-diphosphate-ribose) polymerase-1

Zhu

Martinvalet

Chowdhury

et al. 2009

Blood

Self Cite

View full text Add to dashboard Cite

Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System

Cited by 40 publications

References 31 publications

HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A

Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis

The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5′-diphosphate-ribose) polymerase-1

Contact Info

Product

Resources

About