Roles of PTBP1 in alternative splicing, glycolysis, and oncogensis.

Polypyrimidine tract-binding protein 1 (PTBP1) plays an essential role in splicing and is expressed in almost all cell types in humans, unlike the other proteins of the PTBP family. PTBP1 mediates several cellular processes in certain types of cells, including the growth and differentiation of neuronal cells and activation of immune cells. Its function is regulated by various molecules, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and RNA-binding proteins. PTBP1 plays roles in various diseases, particularly in some cancers, including colorectal cancer, renal cell cancer, breast cancer, and glioma. In cancers, it acts mainly as a regulator of glycolysis, apoptosis, proliferation, tumorigenesis, invasion, and migration. The role of PTBP1 in cancer has become a popular research topic in recent years, and this research has contributed greatly to the formulation of a useful therapeutic strategy for cancer. In this review, we summarize recent findings related to PTBP1 and discuss how it regulates the development of cancer cells.

Polypyrimidine tract-binding proteins are essential for B cell development.

Polypyrimidine tract-binding protein 1 (PTBP1) is a RNA-binding protein (RBP) expressed throughout B cell development. Deletion of Ptbp1 in mouse pro-B cells results in upregulation of PTBP2 and normal B cell development. We show that PTBP2 compensates for PTBP1 in B cell ontogeny as deletion of both Ptbp1 and Ptbp2 results in a complete block at the pro-B cell stage and a lack of mature B cells. In pro-B cells PTBP1 ensures precise synchronisation of the activity of cyclin dependent kinases at distinct stages of the cell cycle, suppresses S-phase entry and promotes progression into mitosis. PTBP1 controls mRNA abundance and alternative splicing of important cell cycle regulators including CYCLIN-D2, c-MYC, p107 and CDC25B. Our results reveal a previously unrecognised mechanism mediated by a RBP that is essential for B cell ontogeny and integrates transcriptional and post-translational determinants of progression through the cell cycle.

Role of polypyrimidine tract-binding protein 1/yin yang 2 signaling in regulating vascular smooth muscle cell proliferation and neointima hyperplasia.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular restenosis. We investigated whether polypyrimidine tract-binding protein 1 (PTBP1), a novel regulator of cell proliferation and differentiation, is implicated in VSMC proliferation and neointima hyperplasia responding to injury. C57BL/6 J mice of 10-12 weeks old were randomly divided into sham and carotid artery injury group. Primary VSMCs obtained from thoracic aortas of 10- to 12-week-old mice were treated with physiological saline and platelet derived growth factor-BB (PDGF-BB). Adenovirus expressing shCon, shPTBP1 or shYY2 were transfected into the injured common carotid artery or VSMCs. qRT-PCR and immunoblotting were used to determine the mRNA and protein expression levels, respectively. Immunohistochemical staining of H&E and Ki-67 were used to evaluate restenosis of vessels. Cell counting kit-8 assay and Ki-67 immunofluorescent staining were utilized to evaluate the rate of VSMC proliferation. The expression of PTBP1 were upregulated both in injured arteries and in PDGF-BB-treated VSMCs. PTBP1 inhibition significantly attenuated neointima hyperplasia and Ki-67 positive area induced by injury. Knockdown of PTBP1 in vitro also suppressed VSMC proliferation after PDGF-BB treatment. The effects of PTBP1 inhibition mentioned above were all abolished by knockdown of YY2. Finally, we identified four cell cycle regulators (p53, p21, Cdkn1c, Cdkn2b) that were regulated by PTBP1/YY2 axis both in vitro and in vivo. These findings demonstrated that PTBP1 is a critical regulator of VSMC proliferation and neointima hyperplasia via modulating the expression of YY2.

PTBP1 contributes to spermatogenesis through regulation of proliferation in spermatogonia.

Polypyrimidine tract-binding protein 1 (PTBP1) is a highly conserved RNA-binding protein that is a well-known regulator of alternative splicing. Testicular tissue is one of the richest tissues with respect to the number of alternative splicing mRNA isoforms, but the molecular role(s) of PTBP1 in the regulation of these isoforms during spermatogenesis is still unclear. Here, we developed a germ cell-specific Ptbp1 conditional knockout (cKO) mouse model by using the Cre-loxP system to investigate the role of PTBP1 in spermatogenesis. Testis weight in Ptbp1 cKO mice was comparable to that in age-matched controls until 3 weeks of age; at ≥ 2 months old, testis weight was significantly lighter in cKO mice than in age-matched controls. Sperm count in Ptbp1 cKO mice at 2 months old was comparable to that in controls, whereas sperm count significantly decreased at 6 months old. Seminiferous tubules that exhibited degeneration in spermatogenic function were more evident in the 2-month-old Ptbp1 cKO mice than in controls. In addition, the early neonatal proliferation of spermatogonia, during postnatal days 1-5, was significantly retarded in Ptbp1 cKO mice compared with that in controls. An in vitro spermatogonia culture model (germline stem cells) revealed that hydroxytamoxifen-induced deletion of PTBP1 from germline stem cells caused severe proliferation arrest accompanied by an increase of apoptotic cell death. These data suggest that PTBP1 contributes to spermatogenesis through regulation of spermatogonia proliferation.

A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival.

Functions of many long noncoding RNAs (lncRNAs) depend on their ability to interact with multiple copies of specific RNA-binding proteins (RBPs). Here, we devised a workflow combining bioinformatics and experimental validation steps to systematically identify RNAs capable of multivalent RBP recruitment. This uncovered a number of previously unknown transcripts encoding high-density RBP recognition arrays within genetically normal short tandem repeats. We show that a top-scoring hit in this screen, lncRNA PNCTR, contains hundreds of pyrimidine tract-binding protein (PTBP1)-specific motifs allowing it to sequester a substantial fraction of PTBP1 in a nuclear body called perinucleolar compartment. Importantly, PNCTR is markedly overexpressed in a variety of cancer cells and its downregulation is sufficient to induce programmed cell death at least in part by stimulating PTBP1 splicing regulation activity. This work expands our understanding of the repeat-containing fraction of the human genome and illuminates a novel mechanism driving malignant transformation of cancer cells.

PTBP1 promotes tumorigenesis by regulating apoptosis and cell cycle in colon cancer.

Increased expression of polypyrimidine tract-binding protein 1 (PTBP1) has been observed in human ovarian tumors, glioblastomas, and breast cancer, but its biological roles in tumorigenesis is not fully clear. In the present research, we investigated the biological role of PTBP1 in colon cancer. We found that PTBP1 was overexpressed both in colon cancer cell lines and tissues. Tissue microarray analysis (TMA) indicated that low PTBP1 expression predicted a favorable overall survival for colon cancer patients. Using small interfering RNA technology, we found that down-regulation of PTBP1 significantly inhibited colon cancer cell growth/proliferation, and induced cell cycle arrest as well as apoptosis in vitro. Western blot analysis showed that siRNA PTBP1 could up-regulate the expression of cytoC, p53 and Bax as well as down-regulated p85, p-AKT, cyclinD1, CDK4 and Bcl2 compared to the control. Furthermore, Caspase-3 and PARP1 were activated when PTBP1 is knockdown. This study implies that PTBP1 plays an important role in tumorigenesis of colon cancer.

Roles of Albumin-Binding Proteins in Cancer Progression and Biomimetic Targeted Drug Delivery.

Nutrient transporters have attracted significant attention for their promising application in biomimetic delivery. Due to the active consumption of nutrients, cancer cells generally overexpress nutrient transporters to meet their increased need for energy and materials. For example, albumin-binding proteins (ABPs) are highly overexpressed in malignant cells, stromal cells, and tumor vessel endothelial cells responsible for albumin uptake. ABP (e.g., SPARC) is a promising target for tumor-specific drug delivery, and albumin has been widely used as a biomimetic delivery carrier. Apart from the transportation function, ABPs are closely associated with neoplasia, invasion, and metastasis. Herein, a summary of the roles of ABP in cancer progression and the application of albumin-based biomimetic tumor-targeted delivery through the ABP pathway is presented.

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis.

Regulation of alternative splicing is an important process for cell differentiation and development. Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exon-centric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed. We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

A mechanism underlying position-specific regulation of alternative splicing.

Many RNA-binding proteins including a master regulator of splicing in developing brain and muscle, polypyrimidine tract-binding protein 1 (PTBP1), can either activate or repress alternative exons depending on the pre-mRNA recruitment position. When bound upstream or within regulated exons PTBP1 tends to promote their skipping, whereas binding to downstream sites often stimulates inclusion. How this switch is orchestrated at the molecular level is poorly understood. Using bioinformatics and biochemical approaches we show that interaction of PTBP1 with downstream intronic sequences can activate natural cassette exons by promoting productive docking of the spliceosomal U1 snRNP to a suboptimal 5' splice site. Strikingly, introducing upstream PTBP1 sites to this circuitry leads to a potent splicing repression accompanied by the assembly of an exonic ribonucleoprotein complex with a tightly bound U1 but not U2 snRNP. Our data suggest a molecular mechanism underlying the transition between a better-known repressive function of PTBP1 and its role as a bona fide splicing activator. More generally, we argue that the functional outcome of individual RNA contacts made by an RNA-binding protein is subject to extensive context-specific modulation.

Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis.

PURPOSE: In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. METHODS: Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. RESULTS: Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. CONCLUSION: Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

Apolipoprotein A1 and heterogeneous nuclear ribonucleoprotein E1 implicated in the regulation of embryo implantation by inhibiting lipid peroxidation.

It is known that apolipoprotein A1 (apoA1) is a stimulator of endothelial nitric oxide synthase (eNOS), and that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1)-containing RNP complexes is a key protector of basal stabilization of eNOS mRNA. Recently, we found that apoA1 and hnRNP-E1 were up-regulated during peri-implantation period, and the purpose of this study was to explore the roles of apoA1 and hnRNP-E1 during this period in the mouse. It was found that the up-regulation of apoA1 and hnRNP-E1 were dependent on the presence and status of blastocysts, on endometrial decidualization and on the progesterone and 17ß-oestradiol status. Knockdown of apoA1 or hnRNP-E1 both resulted in reduced numbers of embryo implantations and neonates (P < 0.01), and lipid peroxidation was found to be involved. On pregnancy day 5 eNOS expression and superoxidase dismutase (SOD) quantity were increased, and malondialdehyde (MDA) quantity was decreased at implantation sites. The knockdown of either apoA1 or hnRNP-E1 led to down-regulation of eNOS (P < 0.01) and to an increase in the quantity of MDA (P < 0.05), and a decrease in the amount of SOD (P < 0.01). These findings suggest that apoA1 and hnRNP-E1 may play roles in embryo implantation by inhibiting lipid peroxidation.

Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first-line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing (AS) of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence-free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favor the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, upregulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to upregulation of PTBP1 and modulation of PKM AS in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy.

Translational regulation of inhibin ßA by TGFß via the RNA-binding protein hnRNP E1 enhances the invasiveness of epithelial-to-mesenchymal transitioned cells.

The epithelial-to-mesenchymal transition (EMT) is a cellular process that functions during embryonic development and tissue regeneration, thought to be aberrantly activated in epithelial-derived cancer and has an important role in the process of metastasis. The transforming growth factor (TGF)-ß signaling pathway is a key inducer of EMT and we have elucidated a posttranscriptional mechanism by which TGFß modulates expression of select transcripts via the RNA-binding protein hnRNP E1 during EMT. One such transcript inhibin ßA is a member of the TGFß superfamily. Here, we show by polysome profiling that inhibin ßA is translationally regulated by TGFß via hnRNP E1. TGFß treatment or knockdown of hnRNP E1 relieves silencing of the inhibin ßA transcript, resulting in increased protein expression and secreted levels of the inhibin ßA homodimer, activin A. Our data indicate that the translational upregulation of inhibin ßA enhances the migration and invasion of cells that have undergone an EMT and promotes cancer progression in vivo.

[RNA-binding protein PTB in spermatogenesis: Progress in studies].

RNA binding proteins (RBPs) regulate the function of cells by interacting with nascent transcripts and therefore are receiving increasing attention from researchers for their roles in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. Further investigations on the post-transcriptional regulation mechanisms and isoforms of PTB proteins in the spermatogenesis show that PTB protein 1 (Ptbp1) is a predominant isoform in mitotic cells (spermatogonia), while Ptbp2 predominates in meiotic spermatocytes and postmeiotic spermatids and binds to the specific 3' untranslated region (3' UTR) of the phosphoglycerate kinase 2 (Pgk-2) mRNA, which helps to stabilize Pgk-2 mRNA in male mouse germ cells. In case of Ptbp2 inactivation in the testis, the differentiation of germ cells arrests in the stage of round spermatids, with proliferation of multinucleated cells in the seminiferous tubule, increased apoptosis of spermatocytes, atrophy of seminiferous tubules, and lack of elongating spermatids, which consequently affects male fertility. This article presents an overview on the structure of the PTB protein and its role in regulating mammalian spermatogenesis.

DNA polymerase η is regulated by poly(rC)-binding protein 1 via mRNA stability.

POLH (DNA polymerase η), a target of p53 tumour suppressor, plays a key role in TLS (translesion DNA synthesis). Loss of POLH is responsible for the human cancer-prone syndrome XPV (xeroderma pigmentosum variant). Owing to its critical role in DNA repair and genome stability, POLH expression and activity are regulated by multiple pathways. In the present study, we found that the levels of both POLH transcript and protein were decreased upon knockdown of the transcript encoding PCBP1 [poly(rC)-binding protein 1]. We also found that the half-life of POLH mRNA was markedly decreased upon knockdown of PCBP1. Moreover, we found that PCBP1 directly bound to the POLH 3'-UTR and the PCBP1-binding site in POLH mRNA is an atypical AU-rich element. Finally, we showed that the AU-rich element in POLH 3'-UTR was responsive to PCBP1 and sufficient for PCBP1 to regulate POLH expression. Taken together, we uncovered a novel mechanism by which POLH expression is controlled by PCBP1 via mRNA stability.

Emerging roles for hnRNPs in post-transcriptional regulation: what can we learn from flies?

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a highly conserved family of RNA-binding proteins able to associate with nascent RNAs in order to support their localization, maturation and translation. Research over this last decade has remarked the importance of gene regulatory processes at post-transcriptional level, highlighting the emerging roles of hnRNPs in several essential biological events. Indeed, hnRNPs are key factors in regulating gene expression, thus, having a number of roles in many biological pathways. Moreover, failure of the activities catalysed by hnRNPs affects various biological processes and may underlie several human diseases including cancer, diabetes and neurodegenerative syndromes. In this review, we summarize some of hnRNPs' roles in the model organism Drosophila melanogaster, particularly focusing on their participation in all aspects of post-transcriptional regulation as well as their conserved role and involvement in the aetiology of human pathologies.

SchA-p85-FAK complex dictates isoform-specific activation of Akt2 and subsequent PCBP1-mediated post-transcriptional regulation of TGFß-mediated epithelial to mesenchymal transition in human lung cancer cell line A549.

A post-transcriptional pathway by which TGF-ß modulates expression of specific proteins, Disabled-2 (Dab2) and Interleukin-like EMT Inducer (ILEI), inherent to epithelial to mesenchymal transition (EMT) in murine epithelial cells through Akt2-mediated phosphorylation of poly r(C) binding protein (PCBP1), has been previously elucidated. The aims of the current study were to determine if the same mechanism is operative in the non-small cell lung cancer (NSCLC) cell line, A549, and to delineate the underlying mechanism. Steady-state transcript and protein expression levels of Dab2 and ILEI were examined in A549 cells treated with TGF-ß for up to 48 h. Induction of translational de-repression in this model was quantified by polysomal fractionation followed by qRT-PCR. The underlying mechanism of isoform-specific activation of Akt2 was elucidated through a combination of co-immunoprecipitation studies. TGF-ß induced EMT in A549 cells concomitant with translational upregulation of Dab2 and ILEI proteins through isoform-specific activation of Akt2 followed by phosphorylation of PCBP1 at serine-43. Our experiments further elucidated that the adaptor protein SchA is phosphorylated at tyrosine residues following TGF-ß treatment, which initiated a signaling cascade resulting in the sequential recruitment of p85 subunit of PI3K and focal adhesion kinase (FAK). The SchA-FAK-p85 complex subsequently selectively recruited and activated Akt2, not Akt1. Inhibition of the p85 subunit through phosphorylated 1257 peptide completely attenuated EMT in these cells. We have defined the underlying mechanism responsible for isoform-specific recruitment and activation of Akt2, not Akt1, during TGF-ß-mediated EMT in A549 cells. Inhibition of the formation of this complex thus represents an important and novel therapeutic target in metastatic lung carcinoma.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) and human transformer-2-beta1 (hTra2-beta1)-regulated estrogen receptor-alpha improves prognosis of endometrial cancer.

PURPOSE OF INVESTIGATION: Heterogeneous nuclear ribonucleoprotein (hnRNP) family possesses decreasing effect towards endometrial cancer (EC) and human transformer-2-betal (hTra2-betal) performs an intimate relationship with EC, either. Recent study shows that hnRNPs and hTra2-betal regulate the genetic expression, which is concerned with estrogen receptor (ER). MATERIALS AND METHODS: The present study was designed to investigate the link between ER and hnRNPs or hTra2-betal in the prognosis of EC patients by Real-time PCR and immunohistochemisty (IHC). RESULTS: Results showed that ER protein expression presented a significant change in the recurrence and outcome of EC patients, and the nucleus hTra2-betal protein expression was also increased in the recurrent patients, indicating that the three might be important in ER expression in the prognosis therapy of EC patients. CONCLUSION: The present findings provide an insight of pharmaceutical targeting therapy and prognosis of EC.

Poly (C)-binding protein 1 regulates p63 expression through mRNA stability.

p63, a transcription factor and p53 family protein, plays a crucial role in tumor suppression and development of various epithelial tissues. While p63 expression is controlled mostly by post-translational modifications, recent studies indicate that transcriptional and posttranscriptional regulations are essential for proper p63 expression. Here, we investigated the regulation of p63 expression by poly (C)-binding protein 1 (PCBP1, also known as hnRNP-E1 and αCP1). We found that knockdown of PCBP1 decreases the level of p63 transcript and protein. We also found that PCBP1 regulates the stability of p63 mRNA via binding to p63 3'UTR. Additionally, we found that a CU-rich element (CUE) in p63 3'UTR is bound by and responsive to PCBP1. Together, we conclude that PCBP1 regulates p63 expression via mRNA stability.

Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin.

The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.