Identification of an intrinsic determinant critical for maspin subcellular localization and function.

Sijana H Dzinic,David Krass,Fulvio Lonardo,Margarida Bernardo,Paul Stemmer,Xiaohua Li,Ivory Dean,Namhee Shin,Yonghong Meng,Shijie Sheng,Alexander Kaplun,Hari K Koul

doi:10.1371/journal.pone.0074502

Sijana H Dzinic, David Krass + Show 10 more

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https://doi.org/10.1371/journal.pone.0074502

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Abstract

Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1). Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL) and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate346 (D346) to Glutamate (E346), maspinD346E, was predominantly nuclear, whereas wild type maspin (maspinWT) was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC) showed that the increased nuclear enrichment of maspinD346E was, at least in part, due to its increased affinity to HDAC1. MaspinD346E was also more potent than maspinWT as an HDAC inhibitor. Taken together, our evidence demonstrates that D346 is a critical cis-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.

Highlights

Tumor suppressor maspin is a member of Clade B serine protease inhibitor superfamily, but has been shown to have deviant biological functions and molecular modes of action [1,2]
It was noted that when used at the same multiplicity of infection (MOI), adenoviruses encoding different maspin mutants resulted in an unequal level of protein production (Figure 1C), with maspinWT expressed at the highest level, followed by the maspinD346E
We modified the infection MOI for each mutant to equalize the level of protein expression and made sure that the level of maspinWT or maspinD346E expressed in infected DU145 cells did not exceed the level of maspin endogenously expressed by normal immortalized prostate epithelial cells CRL2221 (Figure 1D)

Summary

Introduction

Tumor suppressor maspin is a member of Clade B serine protease inhibitor (serpin) superfamily, but has been shown to have deviant biological functions and molecular modes of action [1,2]. Our laboratory discovered that endogenous maspin binds to and inhibits the activity of histone deacetylase 1 (HDAC1) [7,9], which is a major nuclear deacetylase of class I that is up-regulated in many types of cancers [2]. As compared to pharmacological HDAC inhibitors, maspin regulates a much smaller cluster of HDAC target genes, all being implicated in controlling epithelial differentiation [12]. These findings provided a functional connection between maspin and a better differentiated phenotype as well as better prognosis of human cancer [13]

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