Identification of a Nuclear Targeting Domain in the Insertion between Helices C and D in Protease Inhibitor-10

Chuang, Trinette; Schleef, Raymond R.

doi:10.1074/jbc.274.16.11194

Cited by 34 publications

(38 citation statements)

References 31 publications

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“…A remarkable finding was the prominent nuclear localization of PI-8, particularly in epithelial cells. Although nuclear localization of intracellular serpins has recently been described (Chuang and Schleef 1999;Bird et al 2001), our study is, as far as we know, the first to describe nuclear localization of an endogenous serpin in human tissues.…”

Section: Discussionmentioning

confidence: 99%

“…Until recently, intracellular serpins were considered to reside only in the cytoplasm, although some, such as PAI-2, are partially secreted. As a first exception, PI-10 was shown to have an insertion between the helices C and D, which contains an NLS (Chuang and Schleef 1999). do not contain such an insertion and also do not contain a conventional NLS.…”

Section: Discussionmentioning

confidence: 99%

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Distribution of the Human Intracellular Serpin Protease Inhibitor 8 in Human Tissues

Strik

Bladergroen

Wouters

et al. 2002

J Histochem Cytochem.

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S U M M A R YOvalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P 1 residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Distribution of the Human Intracellular Serpin Protease Inhibitor 8 in Human Tissues

Strik

Bladergroen

Wouters

et al. 2002

J Histochem Cytochem.

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“…Washed cells were disrupted with a Dounce homogenizer, and nuclei were isolated as described previously (3). The cytosol-containing supernatant above the nuclear pellet was harvested and the clarified by centrifugation (15,000 ϫ g, 30 min).…”

Section: Methodsmentioning

confidence: 99%

“…Transfection and Fractionation of HeLa Cells-Transfection and selection of HeLa cells with mammalian expression vectors were performed as described previously (3). Washed cells were disrupted with a Dounce homogenizer, and nuclei were isolated as described previously (3).…”

Section: Methodsmentioning

confidence: 99%

Protease Inhibitor 10 Inhibits Tumor Necrosis Factor α-induced Cell Death

Schleef

Chuang

2000

Journal of Biological Chemistry

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Protease inhibitor 10 (PI10) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that is expressed at elevated levels in patients with acute myeloid leukemia and chronic myelomonocytic leukemia. Based upon the ability of the related serpin plasminogen activator inhibitor 2 (PAI-2) to protect cells against tumor necrosis factor ␣ (TNF␣)-induced cell death, this study was initiated to investigate the potential cytoprotective activity of PI10. Two different expression systems (i.e. plasmids encoding either PI10 alone or PI10 fused to the tag: enhanced green fluorescent protein, EGFP) were utilized to stably transfect an eukaryotic model cell system (i.e. HeLa cells) that neither expresses PAI-2 nor PI10. The level of PI10 expression in the stable transfectants was found to correlate with their resistance to TNF␣-induced cell death. Immunoprecipitation/immunoblotting experiments demonstrated that PI10 is able to form SDS-stable complexes (i.e. M r >100,000) with a cytosolic protein(s). Increased levels of the PI10-containing complexes can be detected by TNF␣ treatment by preventing intracellular degradative activities with the proteasome inhibitor N-carbobenzyloxy-leucine-leucine-norvalinal. PI10-containing complexes are dissociated with conditions known to separate classical protease-serpin complexes (i.e., 1.5 M ammonium hydroxide in the presence of SDS). These data support a role for the regulation of intracellular protease activities by ov-serpins. Protease inhibitor 10 (PI10)1 (1) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that exhibits an expression pattern restricted to cells within the monocytic lineage (2). The absence of a signal leader sequence in PI10's coding sequence (1, 2) coupled with a nuclear localization signal in its C-D interhelical loop (3) results in this inhibitor being present both as a cytoplasmic and a nuclear molecule. The unique tissue and subcellular distribution for this serpin raises the possibility that PI10 may play a key role in normal hematopoietic development, a process that requires the regulation of cell viability, growth, and differentiation.The ability of diverse types of serine protease inhibitors to prevent cell death suggests that intracellular proteases other than cysteine aspartic-specific proteases can also participate in this process (for reviews, see Refs. 4 and 5). This concept is supported by the observation that the ov-serpin PAI-2 is able to confer resistance from the cytotoxic effects of tumor necrosis factor ␣ (TNF␣) in two cell lines (i.e. HT1080 (6) and HeLa (7)). Unfortunately, high M r SDS-stable complexes between PAI-2 and another molecule have not been observed during these cell death experiments, thus impeding further research on the pathway. Based upon (i) the high amino acid homology (48%) between PAI-2 and PI110 (1) and (ii) our observations that patients with acute myeloid leukemia and chronic myelomonocytic leukemia express high levels of PI10 (2), we hypothesized that this ov-serpin might ...

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“…placental microvillous membranes) without affecting inhibitory activity (Jensen et al 1993). Several other serpins, such as protease inhibitor-10 (PI 10) and myeloid/erythroid nuclear termination stage-specific protein (MENT) also contain extended loops between helices C and D which act as nuclear targeting signals , Chuang & Schleef 1999. Image generated by Swiss-PdbViewer V3.5b3 (Guex et al 1997); P D B ID# 1 B Y 7 (Harrop et al 1999).…”

Section: Protein Structure Of Pai-2mentioning

confidence: 99%

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

1998

Fibrinolysis and Proteolysis

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Identification of a Nuclear Targeting Domain in the Insertion between Helices C and D in Protease Inhibitor-10

Cited by 34 publications

References 31 publications

Distribution of the Human Intracellular Serpin Protease Inhibitor 8 in Human Tissues

Distribution of the Human Intracellular Serpin Protease Inhibitor 8 in Human Tissues

Protease Inhibitor 10 Inhibits Tumor Necrosis Factor α-induced Cell Death

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

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