Jens Berthelsen

The novel homeoprotein Prep1 modulates Pbx–Hox protein cooperativity

The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1–Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N‐terminal portions of both proteins. The Prep1–Pbx protein–protein interaction is essential for DNA‐binding activity. Prep1–Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1–Pbx1–HOXB1 complex on a HOXB1‐responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1–Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

Prep1, a novel functional partner of Pbx proteins

The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. We reported previously that purified UEF3 is a complex of several subunits. In this paper we report the cloning of the cDNA of one of the subunits which encodes for a novel human homeodomain protein, which we have termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains, is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. We further identify the other UEF3 subunit as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity towards the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo.

Direct interaction of two homeoproteins, homothorax and extradenticle, is essential for EXD nuclear localization and function.

The Drosophila Homothorax (HTH) and Extradenticle (EXD) are two homeoproteins required in a number of developmental processes. EXD can function as a cofactor to Hox proteins. Its nuclear localization is dependent on HTH. In this study we present evidence of in vivo physical interaction between HTH and EXD, mediated primarily through an evolutionarily conserved MH domain in HTH. This interaction is essential for the mutual stabilization of both proteins, for EXD nuclear localization, and for the cooperative DNA binding of the EXD-HTH heterodimer. Some in vivo functions require both EXD and HTH in the nucleus, suggesting that the EXD-HTH complex may function as a transcriptional regulator.

The PBX-Regulating Protein PREP1 is present in different PBX-complexed forms in mouse

Human PREP1, a novel homeodomain protein of the TALE super-family, forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters (Berthelsen, J., Zappavigna, V., Ferretti, E., Mavilio, F., Blasi, F. , 1998b. The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperatity. EMBO J. 17, 1434-1445; Berthelsen, J., Zappavigna, V., Mavilio, F., Blasi, F., 1998c. Prep1, a novel functional partner of Pbx proteins. EMBO J. 17, 1423-1433). Here we demonstrate the presence of DNA-binding PREP1-PBX complexes also in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. We report the cloning and expression domain of murine Prep1 gene. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. We further demonstrate the presence of murine Prep1 mRNA and protein, and of different DNA-binding PREP1-PBX complexes, in mouse embryos from at least 9.5 days p.c. Moreover, we show that PREP1 is present in all embryonic tissues from at least 7.5-17.5 days p.c with a predominantly nuclear staining. PREP1 is able to super-activate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and we show that all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role of PREP1 as a regulator of PBX and HOXB-1 proteins activity during development.

A secretory system for bacterial production of high-profile protein targets

Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N‐terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high‐throughput compatible system, we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.

A Comparison of Protein Kinases Inhibitor Screening Methods Using Both Enzymatic Activity and Binding Affinity Determination

Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits.

Analogues of the Natural Product Sinefungin as Inhibitors of EHMT1 and EHMT2.

A series of analogues of the natural product sinefungin lacking the amino acid moiety was synthesized and probed for their ability to inhibit EHMT1 and EHMT2. This study led to inhibitors 3b and 4d of methyltransferase activity of EHMT1 and EHMT2 and it demonstrates that such analogues constitute an interesting scaffold to develop selective methyltransferase inhibitors. Surprisingly, the inhibition was not competitive toward AdoMet.

The LapG protein plays a role in Pseudomonas aeruginosa biofilm formation by controlling the presence of the CdrA adhesin on the cell surface

Pseudomonas aeruginosa is a clinically relevant species involved in biofilm‐based chronic infections. We provide evidence that the P. aeruginosa LapG protein functions as a periplasmic protease that can cleave the protein adhesin CdrA off the cell surface, and thereby plays a role in biofilm formation and biofilm dispersal. The P. aeruginosa LapG protein is shown to be a functional homolog of the Pseudomonas putida LapG protein which has previously been shown to function as a periplasmic protease that targets the surface adhesin LapA. Transposon mutagenesis and characterization of defined knockout mutants provided evidence that the CdrA adhesin is a target of LapG in P. aeruginosa. A wspF lapG double mutant was hyper‐aggregating and hyper biofilm forming, whereas a wspF lapG cdrA triple mutant lost these phenotypes. In addition, western blot detection of CdrA in culture supernatants and whole‐cell protein fractions showed that CdrA was retained in the whole‐cell protein fraction when LapG was absent, whereas it was found in the culture supernatant when LapG was present. The finding that CdrA is a target of LapG in P. aeruginosa is surprising because CdrA has no homology to LapA.

Conjugation of Cell-Penetrating Peptides to Parathyroid Hormone Affects Its Structure, Potency, and Transepithelial Permeation

Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid hormone, i.e., PTH(1-34), and to evaluate the effect with regard to secondary structure, potency in Saos-2 cells, immunogenicity, safety, as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative to covalent conjugation was compared with regard to the transepithelial permeation. CPP-conjugated PTH(1-34) fusion peptides were successfully expressed in Escherichia coli and purified from inclusion bodies. No clear correlation between the degree of secondary structure of the CPP-conjugated PTH(1-34) fusion peptides and their potency was found, albeit a general decrease in permeation was observed for both N- and C-terminally CPP-conjugated PTH(1-34) as compared to native PTH(1-34). However, attachment of CPP to the N-terminus significantly increased permeation across Caco-2 cell monolayers as compared to the corresponding C-terminally CPP-conjugated PTH(1-34). In addition, the nonaarginine sequence proved to be the only CPP capable of increasing permeation when conjugated to PTH(1-34) as compared to co-administration of CPP and PTH(1-34). This enhancement effect was, however, associated with an unacceptably low level of cell viability. In conclusion, covalent conjugation of CPPs to PTH(1-34) influenced the secondary structure, potency, and transepithelial permeation efficiency of the resulting conjugate, and hence this approach appears not to be favorable as compared to co-administration when optimizing CPP-mediated permeation of PTH(1-34) across an intestinal epithelium.

B-lymphoid tyrosine kinase (Blk) is an oncogene and a potential target for therapy with dasatinib in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphoma (CTCL) is the most frequent primary lymphoma of the skin. Patients diagnosed in early stages often experience an indolent disease course and have a favorable prognosis. Yet, the disease follows an aggressive course in a substantial fraction (15–20%) of patients and despite recent progress in novel therapies, advanced disease remains a major challenge as relapses are common and cure is rare.1 Recently, it was discovered,2 and independently confirmed in a meta-analysis study,3 that malignant T cells in the majority of patients display ectopic expression of the B-lymphoid tyrosine kinase (Blk), a member of the Src kinase family. Importantly, gene knockdown experiments showed that Blk promoted the proliferation of malignant T cells from CTCL patients,2 suggesting that Blk—in analogy with other Src family members—may function as an oncogene. In support, Montero-Ruiz et al.4 provided evidence that Blk is implicated in childhood acute lymphoblastic leukemia. However, studies in mice suggested that murine Blk also has tumor-suppressive functions depending on the specific cellular context.5 To study the oncogenic potential of human Blk, we therefore transfected a cytokine (IL-3)-dependent lymphoid cell line (Ba/F3) with plasmids expressing either wild-type (wt) Blk or a constitutively active form of Blk lacking the kinase-inhibitory site due to a tyrosine-to-phenylalanine substitution at amino-acid position 501 (Y501F). Stable transfectants were established by selecting for the plasmid-encoded blasticidin resistance gene, and before experimentation, transformed cells were maintained in blasticidin- and IL-3-supplemented growth media. As shown in Figure 1a, the constitutively active form of Blk (Y501F) was fully able to transform growth factor (IL-3)-dependent Ba/F3 cells into IL-3-independent cells, whereas non-transfected and Blk-wt-transfected Ba/F3 cells remained dependent on exogenous IL-3 to survive and proliferate. In accordance, IL-3 deprivation induced massive apoptosis in non-transfected and Blk-wt-transfected Ba/F3 cells, whereas no increase in apoptosis was observed in Blk(Y501F)-transfected Ba/F3 cells following IL-3 withdrawal (Figure 1b). As expected, Blk(Y501F) was phosphorylated on the activating tyrosine (Y388) and not on the inhibitory tyrosine phosphorylation site (Y501), whereas Blk-wt was heavily phosphorylated on the kinase-inhibitory site (Y501) (Figures 1c and d). The well-characterized Src family kinase inhibitor, Lck inhibitor (LckI, Calbiochem, San Diego, CA, USA), selectively inhibited the proliferation of Blk(Y501F)-transfected Ba/F3 cells, whereas an inhibitor of mitogen-activated protein kinase p38 (SB203580, Selleck Chemicals, Houston, TX, USA) did not (Figure 1e). Likewise, the dual-specificity inhibitor of Bcr-Abl and the Src family kinases, dasatinib (Sprycel, Selleck Chemicals), inhibited Y388 phosphorylation and proliferation of the Blk(Y501F)-transfected Ba/F3 cells (Figures 1f and g). Taken together, these results indicate that the active form of human Blk is able and sufficient to transform cytokine-dependent lymphoid cells into cytokine-independent cells. These findings support the previous observation made by others6 that murine Blk(Y495F) is lymphomagenic in mice. In keeping, enzymatic inhibition by LckI and other Src family kinase inhibitors, as well as siRNA-mediated knockdown of Blk, inhibits proliferation of Blk-positive malignant T cells including MyLa2059 and MyLa2000 (ref. 2 and data not shown). As dasatinib profoundly inhibited Blk(Y501F)-transformed Ba/F3 cells and tyrosine phosphorylation of Blk in the MyLa2059 cells (Figure 2a), we hypothesized that dasatinib—which is used for treatment of chronic myelogenous leukemia and other malignancies7—has a potential for treatment of CTCL. To address this hypothesis, we initially studied the effect of dasatinib on malignant proliferation in vitro. As shown in Figure 2b, dasatinib inhibited the spontaneous proliferation of the malignant CTCL T-cell line MyLa2059 in a concentration-dependent manner. Likewise, the Blk-positive CTCL cell lines MyLa2000 and PB2B2 were also inhibited by dasatinib, whereas the Blk-negative Sezary Syndrome cell line (SeAx) was resistant (Supplementary Figure S1). As malignant T cells, including some cell lines obtained from CTCL2 and peripheral T-cell lymphoma patients,8 often express Fyn (another Src kinase), we cannot exclude the possibility that the effect of dasatinib was partially mediated through an inhibition of both Blk and Fyn. However, Fyn is not tyrosine phosphorylated in the malignant MyLa cells suggesting that Fyn may not be functionally active in these cells (data not shown). The observation that dasatinib inhibited Blk-positive tumor cells prompted us to examine the effect of dasatinib on tumor growth in a xenograft transplantation model of CTCL.9, 10 In a preliminary experiment, mice were inoculated subcutaneously (s.c.) with MyLa2059 cells and treated orally with different concentrations of dasatinib (or vehicle as a control) to evaluate the effect on tumor formation in vivo. The average time of tumor onset was significantly (P<0.05) delayed from day 13 in the control group (N=5) to day 20 in animals (N=6) treated with dasatinib (data not shown). Next, we addressed whether dasatinib inhibited growth of already established tumors. Accordingly, eight mice were inoculated s.c. with MyLa2059 cells and following detection of palpable tumors (day 1) the mice were treated with either dasatinib (Sprycel) (40 mg/kg) or vehicle as control. Tumor dimensions were measured in each group on days 3, 5, 8 and 10. As shown in Figure 2c, dasatinib significantly inhibited tumor growth. Likewise, volume of the resected tumors harvested on day 10 was significantly lower in the dasatinib-treated mice when compared with the control mice, confirming that dasatinib does inhibit CTCL tumor growth in vivo (Figure 2d). As the malignant T cells do not express the Bcr-Abl oncogene (data not shown), the present finding suggests that Blk functions as an oncogene in the CTCL cells. As NF-κB is active in CTCL2 and supports growth of dasatinib/imatinib-resistant cells,11, 12 we tested dasatinib in combination with NF-κB inhibitors. Interestingly, dasatinib and NF-κB inhibitors had an additive effect on malignant proliferation in vitro (data not shown), which might explain why Blk(Y501F)-transformed Ba/F3 cells were more sensitive to dasatinib than the malignant MyLa cells. In summary, (i) Blk is enzymatically active in malignant CTCL cells and expressed in situ,2 (ii) its constitutively active form confers cytokine independence (Figure 1) and (iii) it promotes tumor growth in vivo as indicated by the effect of dasatinib on tumor growth in mice (Figure 2). Taken together, these findings strongly suggest that Blk is a potential therapeutic target in CTCL for dasatinib and other clinical-grade dual-specificity Bcr-Abl and Src family kinase inhibitors. As dasatinib and other dual-specific inhibitors are already used in treatment of other hematological malignancies with a high efficacy, tolerability and compliance,7 these drugs are attractive novel candidates for the treatment of CTCL expressing Blk.