An Effective Modification Strategy to Build Multifunctional Peptides Based on a Trypsin Inhibitory Peptide of the Kunitz Family.

Peptides with antimicrobial activity or protease inhibitory activity are potential candidates to supplement traditional antibiotics or cancer chemotherapies. However, the potential of many peptides are limited by drawbacks such as cytotoxicity or susceptibility to hydrolysis. Therefore, strategies to modify the structure of promising peptides may represent an effective approach for developing more promising clinical candidates. In this study, the mature peptide OSTI-1949, a Kunitz-type inhibitor from Odorrana schmackeri, and four designed analogues were successfully synthesised. In contrast to the parent peptide, the analogues showed impressive multi-functionality including antimicrobial, anticancer, and trypsin inhibitory activities. In terms of safety, there were no obvious changes observed in the haemolytic activity at the highest tested concentration, and the analogue OSTI-2461 showed an increase in activity against cancer cell lines without cytotoxicity to normal cells (HaCaT). In summary, through structural modification of a natural Kunitz-type peptide, the biological activity of analogues was improved whilst retaining low cytotoxicity. The strategy of helicity enhancement by forming an artificial α-helix and ß-sheet structure provides a promising way to develop original bioactive peptides for clinical therapeutics.

Cathepsin V regulates cell cycle progression and histone stability in the nucleus of breast cancer cells.

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of the molecular role of the protease within Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome. Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation by MTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment. Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells. Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation.

CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

Exploiting the anticancer effects of a nitrogen bisphosphonate nanomedicine for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an incurable aggressive brain cancer in which current treatment strategies have demonstrated limited survival benefit. In recent years, nitrogen-containing bisphosphonates (N-BPs) have demonstrated direct anticancer effects in a number of tumour types including GBM. In this study, a nano-formulation with the RALA peptide was used to complex the N-BP, alendronate (ALN) into nanoparticles (NPs) < 200 nm for optimal endocytic uptake. Fluorescently labelled AlexaFluor®647 Risedronate was used as a fluorescent analogue to visualise the intracellular delivery of N-BPs in both LN229 and T98G GBM cells. RALA NPs were effectively taken up by GBM where a dose-dependent response was evidenced with potentiation factors of 14.96 and 13.4 relative to ALN alone after 72 h in LN229 and T98G cells, respectively. Furthermore, RALA/ALN NPs at the IC50, significantly decreased colony formation, induced apoptosis and slowed spheroid growth in vitro. In addition, H-Ras membrane localisation was significantly reduced in the RALA/ALN groups compared to ALN or controls, indicative of prenylation inhibition. The RALA/ALN NPs were lyophilised to enhance stability without compromising the physiochemical properties necessary for functionality, highlighting the suitability of the NPs for scale-up and in vivo application. Collectively, these data show the significant potential of RALA/ALN NPs as novel therapeutics in the treatment of GBM.

Cathepsin V suppresses GATA3 protein expression in luminal A breast cancer.

BACKGROUND: Lysosomal cysteine protease cathepsin V has previously been shown to exhibit elevated expression in breast cancer tissue and be associated with distant metastasis. Research has also identified that cathepsin V expression is elevated in tumour tissues from numerous other malignancies, but despite this, there has been limited examination of the function of this protease in cancer. Here we investigate the role of cathepsin V in breast cancer in order to delineate the molecular mechanisms by which this protease contributes to tumourigenesis. METHODS: Lentiviral transductions were used to generate shRNA cell line models, with cell line validation undertaken using RQ-PCR and Western blotting. Phenotypic changes of tumour cell biology were examined using clonogenic and invasion assays. The relationship between GATA3 expression and cathepsin V was primarily analysed using Western blotting. Site-directed mutagenesis was used to generate catalytic mutant and shRNA-resistant constructs to confirm the role of cathepsin V in regulating GATA3 expression. RESULTS: We have identified that elevated cathepsin V expression is associated with reduced survival in ER-positive breast cancers. Cathepsin V regulates the expression of GATA3 in ER-positive breast cancers, through promoting its degradation via the proteasome. We have determined that depletion of cathepsin V results in elevated pAkt-1 and reduced GSK-3ß expression, which rescues GATA3 from proteasomal degradation. CONCLUSIONS: In this study, we have identified that cysteine protease cathepsin V can suppress GATA3 expression in ER-positive breast cancers by facilitating its turnover via the proteasome. Therefore, targeting cathepsin V may represent a potential therapeutic strategy in ER-positive breast cancers, by restoring GATA3 protein expression, which is associated with a more favourable clinical outcome.

Controlled coupling of an ultrapotent auristatin warhead to cetuximab yields a next-generation antibody-drug conjugate for EGFR-targeted therapy of KRAS mutant pancreatic cancer.

BACKGROUND: Antibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC. METHODS: In vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens. RESULTS: Site-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells. CONCLUSIONS: Together, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

Refined construction of antibody-targeted nanoparticles leads to superior antigen binding and enhanced delivery of an entrapped payload to pancreatic cancer cells.

Antibody-targeted nanoparticles have shown exceptional promise as delivery vehicles for anticancer drugs, although manufacturability challenges have hampered clinical progress. These include the potential for uncontrolled and random antibody conjugation, resulting in masked or inactive paratopes and unwanted Fc domain interactions. To circumvent these issues, we show that the interchain disulfide of cetuximab F(ab) may be selectively re-bridged with a strained alkyne handle, to permit 'click' coupling to azide-capped nanoparticles in a highly uniform and oriented manner. When compared to conventional carbodiimide chemistry, this conjugation approach leads to the generation of nanoparticles with a higher surface loading of cetuximab F(ab) and with markedly improved ability to bind to the target epidermal growth factor receptor. Moreover, we show that entrapment of a camptothecin payload within these nanoparticles can enhance drug targeting to antigen-expressing pancreatic cancer cells, resulting in superior cytotoxicity versus the conventional nanoformulation. Collectively, this work highlights the critical need to develop refined methods for the construction of targeted nanoparticles that will accelerate their clinical translation through improved performance and manufacturability.

Design of N-Terminal Derivatives from a Novel Dermaseptin Exhibiting Broad-Spectrum Antimicrobial Activity against Isolates from Cystic Fibrosis Patients.

Dermaseptins are an antimicrobial peptide family widely identified from the skin secretions of phyllomeudusinae frogs. Here, we identify Dermaseptin-PC (DM-PC), from the skin secretion of Phyllomedusa coelestis, and further investigate the properties of this peptide, and a number of rationally designed truncated derivatives. The truncated 19-mer derived from the N-terminus exhibited similar antimicrobial potency when compared to the parent peptide, but the haemolytic effect of this truncated peptide was significantly decreased. Based on previous studies, the charge and hydrophobicity of truncated derivatives can affect the bioactivity of these peptides and thus we designed a 10-mer derivative with an optimised positive charge and a cyclohexylalanine (Cha) at the C-terminus for enhancing the hydrophobicity, DMPC-10A, which retained the antimicrobial activity of the parent peptide. To further investigate the influence of Cha at the C-terminus on activity, it was substituted by alanine (Ala) to generate another derivative, DMPC-10, but this was found to be much less potent. In addition, DM-PC, DMPC-19 and DMPC-10A not only rapidly killed planktonic bacteria isolated from cystic fibrosis (CF) patient, but also effectively eradicated their biofilm matrices.

A Novel Dermaseptin Isolated from the Skin Secretion of Phyllomedusa tarsius and Its Cationicity-Enhanced Analogue Exhibiting Effective Antimicrobial and Anti-Proliferative Activities.

A novel dermaseptin peptide, dermaseptin-PT9 (DPT9), was isolated and identified from Phyllomedusa tarsius by the combination of molecular cloning and LC-MS analysis. Chemically synthesised DPT9 was broadly effective against the tested microorganisms through the disruption of cell membranes and showed weak haemolytic activity towards horse erythrocytes. It also exhibited anti-proliferative effect against various human cancer cells. Moreover, an analogue with enhanced cationicity, K8, 23-DPT9, in which Asp8 and Glu23 were substituted by lysine residues, had a markedly increased antimicrobial effect against all tested microorganisms and disrupted microbial cell membranes. This analogue also showed no haemolysis at its effective antimicrobial concentrations. In addition, K8, 23-DPT9 displayed an enhanced anti-proliferative effect against cancer cells, while displayed weak activity against the normal human cell line, HMEC-1.

Discovery and Rational Design of a Novel Bowman-Birk Related Protease Inhibitor.

Anuran amphibian skin secretions are a rich source of peptides, many of which represent novel protease inhibitors and can potentially act as a source for protease inhibitor drug discovery. In this study, a novel bioactive Bowman-Birk type inhibitory hexadecapeptide of the Ranacyclin family from the defensive skin secretion of the Fukien gold-striped pond frog, Pelophlax plancyi fukienesis, was successfully isolated and identified, named PPF-BBI. The primary structure of the biosynthetic precursor was deduced from a cDNA sequence cloned from a skin-derived cDNA library, which contains a consensus motif representative of the Bowman-Birk type inhibitor. The peptide was chemically synthesized and displayed a potent inhibitory activity against trypsin (Ki of 0.17 µM), as well as an inhibitory activity against tryptase (Ki of 30.73 µM). A number of analogues of this peptide were produced by rational design. An analogue, which substituted the lysine (K) at the predicted P1 position with phenylalanine (F), exhibited a potent chymotrypsin inhibitory activity (Ki of 0.851 µM). Alternatively, a more potent protease inhibitory activity, as well as antimicrobial activity, was observed when P16 was replaced by lysine, forming K16-PPF-BBI. The addition of the cell-penetrating peptide Tat with a trypsin inhibitory loop resulted in a peptide with a selective inhibitory activity toward trypsin, as well as a strong antifungal activity. This peptide also inhibited the growth of two lung cancer cells, H460 and H157, demonstrating that the targeted modifications of this peptide could effectively and efficiently alter its bioactivity.

USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells.

BACKGROUND: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs). METHODS: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed. RESULTS: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib. CONCLUSIONS: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

A novel antimicrobial peptide, Ranatuerin-2PLx, showing therapeutic potential in inhibiting proliferation of cancer cells.

Antimicrobial peptides are a promising resource for developing novel antibiotic and even anticancer drugs. Here, a 28-mer polypeptide, Ranatuerin-2PLx (R2PLx), was identified from lyophilised skin secretions. The chemically synthetic replicates exhibited moderate and broadspectrum antimicrobial effect against various microorganisms including methicillin-resistant Staphylococcus aureus (MRSA, minimal inhibitory concentration = 256 µM). In addition, R2PLx was found to inhibit the proliferation of several tumour cells, especially showing more potent effect on prostate cancer cell, PC-3. The early cell apoptosis was observed in 6 h by Annexin V-FITC/propidium iodide staining, as well as the activation of Caspase-3 at 5 µM peptide concentration. R2PLx may therefore be promising for developing new therapeutic approach for cancer treatment. Moreover, the artificial deficiency of conserved rana-box loop or net positive charge in C-terminal domain notably reduced the biological activities of the truncated and substituted isoforms, respectively, suggesting for maintaining their biological potency of ranatuerin family requires both cysteine-bridged segment and cationincity within the loop domain in C-terminus.

Biological Activities of Cationicity-Enhanced and Hydrophobicity-Optimized Analogues of an Antimicrobial Peptide, Dermaseptin-PS3, from the Skin Secretion of Phyllomedusa sauvagii.

The skin secretions of the subfamily Phyllomedusinae have long been known to contain a number of compounds with antimicrobial potential. Herein, a biosynthetic dermaseptin-precursor cDNA was obtained from a Phyllomedusa sauvagii skin secretion-derived cDNA library, and thereafter, the presence of the mature peptide, namely dermaseptin-PS3 (DPS3), was confirmed by LC⁻MS/MS. Moreover, this naturally occurring peptide was utilized to design two analogues, K5, 17-DPS3 (introducing two lysine residues at positions 5 and 17 to replace acidic amino acids) and L10, 11-DPS3 (replacing two neutral amino acids with the hydrophobic amino acid, leucine), improving its cationicity on the polar/unipolar face and hydrophobicity in a highly conserved sequence motif, respectively. The results in regard to the two analogues show that either increasing cationicity, or hydrophobicity, enhance the antimicrobial activity. Also, the latter analogue had an enhanced anticancer activity, with pretreatment of H157 cells with 1 µM L10, 11-DPS3 decreasing viability by approximately 78%, even though this concentration of peptide exhibited no haemolytic effect. However, it must be noted that in comparison to the initial peptide, both analogues demonstrate higher membrane-rupturing capacity towards mammalian red blood cells.

Identification of novel Amurin-2 variants from the skin secretion of Rana amurensis, and the design of cationicity-enhanced analogues.

Rana amurensis is important in Chinese medicine as its skin secretions contain abundant bioactive peptides. Here, we have identified the antimicrobial peptide Amurin-2 and three highly-conserved variants, Amurin-2a, Amurin-2b and Amurin-2c through a combination of molecular cloning and MS/MS fragmentation sequencing. Synthetic replicates of these peptides demonstrate potent antimicrobial activity against S. aureus, whilst some have activity against C.albicans and even resistant bacterial MRSA. Furthermore, two Lys-analogues (K4-Amurin-2 and K11-Amurin-2) were designed to improve the bioactive function and the antimicrobial activity of K4-Amurin-2 against E.coli was enhanced distinctly. In addition, the two modified peptides also showed more potent activity against S. aureus, C. albicans and MRSA strains. Meanwhile, these peptides showed inhibitory effect on the cell viability of several cancer cells. As a result, these structural and functional studies of Amurin-2 variants and analogues could provide insights for future antimicrobial peptide design.

Predictors of Patient Satisfaction in Pediatric Oncology.

OBJECTIVE: To understand key drivers of patient satisfaction in pediatric hematology/oncology. METHODS: The "top-box" scores of patient satisfaction surveys from 4 pediatric hematology/oncology practices were collected from 2012 to 2014 at an integrated Children's Health Network. One item, "Likelihood of recommending practice," was used as the surrogate for overall patient satisfaction, and all other items were correlated to this item. RESULTS: A total of 1244 satisfaction surveys were included in this analysis. The most important predictors of overall patient satisfaction were cheerfulness of practice ( r = .69), wait time ( r = .60), and staff working together ( r = .60). The lowest scoring items were getting clinic on phone, information about delays, and wait time at clinic. CONCLUSION: Families bringing their children for outpatient care in a hematology/oncology practice want to experience a cheerful and collaborative medical team. Wait time at clinic may be a key driver in the overall experience for families with children with cancer. Future work should be directed at using this evidence to drive patient experience improvement processes in pediatric hematology/oncology.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages.

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation. METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1ß response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover. RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1ß release from macrophages, through lysosomal destabilization. IL-1ß secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1ß release was significantly reduced in primary macrophages from ctss(-/-) mice. CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1ß release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Targeting Siglecs with a sialic acid-decorated nanoparticle abrogates inflammation.

Sepsis is the most frequent cause of death in hospitalized patients, and severe sepsis is a leading contributory factor to acute respiratory distress syndrome (ARDS). At present, there is no effective treatment for these conditions, and care is primarily supportive. Murine sialic acid-binding immunoglobulin-like lectin-E (Siglec-E) and its human orthologs Siglec-7 and Siglec-9 are immunomodulatory receptors found predominantly on hematopoietic cells. These receptors are important negative regulators of acute inflammatory responses and are potential targets for the treatment of sepsis and ARDS. We describe a Siglec-targeting platform consisting of poly(lactic-co-glycolic acid) nanoparticles decorated with a natural Siglec ligand, di(α2→8) N-acetylneuraminic acid (α2,8 NANA-NP). This nanoparticle induced enhanced oligomerization of the murine Siglec-E receptor on the surface of macrophages, unlike the free α2,8 NANA ligand. Furthermore, treatment of murine macrophages with these nanoparticles blocked the production of lipopolysaccharide-induced inflammatory cytokines in a Siglec-E-dependent manner. The nanoparticles were also therapeutically beneficial in vivo in both systemic and pulmonary murine models replicating inflammatory features of sepsis and ARDS. Moreover, we confirmed the anti-inflammatory effect of these nanoparticles on human monocytes and macrophages in vitro and in a human ex vivo lung perfusion (EVLP) model of lung injury. We also established that interleukin-10 (IL-10) induced Siglec-E expression and α2,8 NANA-NP further augmented the expression of IL-10. Indeed, the effectiveness of the nanoparticle depended on IL-10. Collectively, these results demonstrated a therapeutic effect of targeting Siglec receptors with a nanoparticle-based platform under inflammatory conditions.

USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor.

Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.

A novel RCE1 isoform is required for H-Ras plasma membrane localization and is regulated by USP17.

Processing of the 'CaaX' motif found on the C-termini of many proteins, including the proto-oncogene Ras, requires the ER (endoplasmic reticulum)-resident protease RCE1 (Ras-converting enzyme 1) and is necessary for the proper localization and function of many of these 'CaaX' proteins. In the present paper, we report that several mammalian species have a novel isoform (isoform 2) of RCE1 resulting from an alternate splice site and producing an N-terminally truncated protein. We demonstrate that both RCE1 isoform 1 and the newly identified isoform 2 are required to reinstate proper H-Ras processing and thus plasma membrane localization in RCE1-null cells. In addition, we show that the deubiquitinating enzyme USP17 (ubiquitin-specific protease 17), previously shown to modulate RCE1 activity, can regulate the abundance and localization of isoform 2. Furthermore, we show that isoform 2 is ubiquitinated on Lys43 and deubiquitinated by USP17. Collectively, the findings of the present study indicate that RCE1 isoform 2 is required for proper 'CaaX' processing and that USP17 can regulate this via its modulation of RCE1 isoform 2 ubiquitination.

A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity.

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be "fit for purpose" for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients.