Bacterial DnaK reduces the activity of anti-cancer drugs cisplatin and 5FU.

BACKGROUND: Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. METHODS: To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells' survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. RESULTS: Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. CONCLUSIONS: Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.

A heterodimer of α and ß hemoglobin chains functions as an innate anticancer agent.

Metastatic (as well as tumor) microenvironments contain both cancer-promoting and cancer-restraining factors. The balance between these opposing forces determines the fate of cancer cells that disseminate to secondary organ sites. In search for microenvironmental drivers or inhibitors of metastasis, we identified, in a previous study, the beta subunit of hemoglobin (HBB) as a lung-derived antimetastatic factor. In the present study, exploring mechanisms regulating melanoma brain metastasis, we discovered that brain-derived factors restrain proliferation and induce apoptosis and necrosis of brain-metastasizing melanoma cells. Employing various purification procedures, we identified a heterodimer composed of hemoglobin alpha and beta chains that perform these antimetastatic functions. Neither the alpha nor the beta subunit alone was inhibitory. An alpha/beta chain dimer chemically purified from human hemoglobin inhibited the cell viability of primary melanomas, melanoma brain metastasis (MBM), and breast cancer cell lines. The dimer-induced DNA damage, cell cycle arrest at the SubG1 phase, apoptosis, and significant necrosis in four MBM cell lines. Proteomic analysis of dimer-treated MBM cells revealed that the dimer downregulates the expression of BRD4, GAB2, and IRS2 proteins, playing crucial roles in cancer cell sustainability and progression. Thus, we hypothesize that the hemoglobin dimer functions as a resistance factor against brain-metastasizing cancer cells.

Flagella-driven motility is a target of human Paneth cell defensin activity.

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Characterization of the interactome profiling of Mycoplasma fermentans DnaK in cancer cells reveals interference with key cellular pathways.

Chaperone proteins are redundant in nature and, to achieve their function, they bind a large repertoire of client proteins. DnaK is a bacterial chaperone protein that recognizes misfolded and aggregated proteins and drives their folding and intracellular trafficking. Some Mycoplasmas are associated with cancers, and we demonstrated that infection with a strain of Mycoplasma fermentans isolated in our lab promoted lymphoma in a mouse model. Its DnaK is expressed intracellularly in infected cells, it interacts with key proteins to hamper essential pathways related to DNA repair and p53 functions and uninfected cells can take-up extracellular DnaK. We profile here for the first time the eukaryotic proteins interacting with DnaK transiently expressed in five cancer cell lines. A total of 520 eukaryotic proteins were isolated by immunoprecipitation and identified by Liquid Chromatography Mass Spectrometry (LC-MS) analysis. Among the cellular DnaK-binding partners, 49 were shared between the five analyzed cell lines, corroborating the specificity of the interaction of DnaK with these proteins. Enrichment analysis revealed multiple RNA biological processes, DNA repair, chromatin remodeling, DNA conformational changes, protein-DNA complex subunit organization, telomere organization and cell cycle as the most significant ontology terms. This is the first study to show that a bacterial chaperone protein interacts with key eukaryotic components thus suggesting DnaK could become a perturbing hub for the functions of important cellular pathways. Given the close interactions between bacteria and host cells in the local microenvironment, these results provide a foundation for future mechanistic studies on how bacteria interfere with essential cellular processes.

Human intelectin-1 (ITLN1) genetic variation and intestinal expression.

Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan ß-D-galactofuranose or protein-protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.

Mechanism through Which Retrocyclin Targets Flavivirus Multiplication.

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

Human Enteric Defensin 5 Promotes Shigella Infection of Macrophages.

Human α-defensins are 3- to 5-kDa disulfide-bridged peptides with a multitude of antimicrobial activities and immunomodulatory functions. Recent studies show that human enteric α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, aids the highly infectious enteropathogen Shigella in breaching the intestinal epithelium in vitro and in vivo Whether and how HD5 influences Shigella infection of resident macrophages following its invasion of the intestinal epithelium remain poorly understood. Here, we report that HD5 greatly promoted phagocytosis of Shigella by macrophages by targeting the bacteria to enhance bacterium-to-cell contacts in a structure- and sequence-dependent fashion. Subsequent intracellular multiplication of phagocytosed Shigella led to massive necrotic cell death and release of the bacteria. HD5-promoted phagocytosis of Shigella was independent of the status of the type 3 secretion system. Furthermore, HD5 neither inhibited nor enhanced phagosomal escape of Shigella Collectively, these findings confirm a potential pathogenic role of HD5 in Shigella infection of not only epithelial cells but also macrophages, illuminating how an enteropathogen exploits a host protective factor for virulence and infection.

Critical determinants of human neutrophil peptide 1 for enhancing host epithelial adhesion of Shigella flexneri.

Human neutrophil peptides (HNPs), also known as human myeloid α-defensins degranulated by infiltrating neutrophils at bacterial infection loci, exhibit broad antomicrobial activities against bacteria, fungi, and viruses. We have made a surprising recent finding that Shigella, a highly contagious, yet poorly adhesive enteric pathogen, exploits human α-defensins including HNP1 to enhance its adhesion to and invasion of host epithelial cells. However, the critical molecular determinants responsible for HNP1-enhanced Shigella adhesion and invasion have yet to be investigated. Using cultured epithelial cells and polarised Caco2 cells as an in vitro infection model, we demonstrated that HNP1 promoted Shigella infection in a structure- and sequence-dependent manner, with two bulky hydrophobic residues, Trp26 and Phe28 important for HNP1 self-assembly, being most critical. The functional importance of hydrophobicity for HNP1-enhanced Shigella infection was further verified by substitutions for Trp26 of a series of unnatural amino acids with straight aliphatic side chains of different lengths. Dissection of the Shigella infection process revealed that bacteria-rather than host cells-bound HNP1 contributed most to the enhancement. Further, mutagenesis analysis of bacterial surface components, while precluding the involvement of lipopolysaccharides (LPS) in the interaction with HNP1, identified outer membrane proteins and the Type 3 secretion apparatus as putative binding targets of HNP1 involved in enhanced Shigella adhesion and invasion. Our findings provide molecular and mechanistic insights into the mode of action of HNP1 in promoting Shigella infection, thus showcasing another example of how innate immune factors may serve as a double-edged sword in health and disease.

Dithiocarbamate-inspired side chain stapling chemistry for peptide drug design.

Two major pharmacological hurdles severely limit the widespread use of small peptides as therapeutics: poor proteolytic stability and membrane permeability. Importantly, low aqueous solubility also impedes the development of peptides for clinical use. Various elaborate side chain stapling chemistries have been developed for α-helical peptides to circumvent this problem, with considerable success in spite of inevitable limitations. Here we report a novel peptide stapling strategy based on the dithiocarbamate chemistry linking the side chains of residues Lys(i) and Cys(i + 4) of unprotected peptides and apply it to a series of dodecameric peptide antagonists of the p53-inhibitory oncogenic proteins MDM2 and MDMX. Crystallographic studies of peptide-MDM2/MDMX complexes structurally validated the chemoselectivity of the dithiocarbamate staple bridging Lys and Cys at (i, i + 4) positions. One dithiocarbamate-stapled PMI derivative, DTCPMI, showed a 50-fold stronger binding to MDM2 and MDMX than its linear counterpart. Importantly, in contrast to PMI and its linear derivatives, the DTCPMI peptide actively traversed the cell membrane and killed HCT116 tumor cells in vitro by activating the tumor suppressor protein p53. Compared with other known stapling techniques, our solution-based DTC stapling chemistry is simple, cost-effective, regio-specific and environmentally friendly, promising an important new tool for the development of peptide therapeutics with improved pharmacological properties including aqueous solubility, proteolytic stability and membrane permeability.

Systematic mutational analysis of human neutrophil α-defensin HNP4.

Defensins are a family of cationic antimicrobial peptides of innate immunity with immunomodulatory properties. The prototypic human α-defensins, also known as human neutrophil peptides 1-3 or HNP1-3, are extensively studied for their structure, function and mechanisms of action, yet little is known about HNP4 - the much less abundant "distant cousin" of HNP1-3. Here we report a systematic mutational analysis of HNP4 with respect to its antibacterial activity against E. coli and S. aureus, inhibitory activity against anthrax lethal factor (LF), and binding activity for LF and HIV-1 gp120. Except for nine conserved and structurally important residues (6xCys, 1xArg, 1xGlu and 1xGly), the remaining 24 residues of HNP4 were each individually mutated to Ala. The crystal structures of G23A-HNP4 and T27A-HNP4 were determined, both exhibiting a disulfide-stabilized canonical α-defensin dimer identical to wild-type HNP4. Unlike HNP1-3, HNP4 preferentially killed the Gram-negative bacterium, a property largely attributable to three clustered cationic residues Arg10, Arg11 and Arg15. The cationic cluster was also important for HNP4 killing of S. aureus, inhibition of LF and binding to LF and gp120. However, F26A, while functionally inconsequential for E. coli killing, was far more deleterious than any other mutations. Similarly, N-methylation of Leu20 to destabilize the HNP4 dimer had little effect on E. coli killing, but significantly reduced the ability of HNP4 to kill S. aureus, inhibit LF, and bind to LF and gp120. Our findings unveil the molecular determinants of HNP4 function, completing the atlas of structure and function relationships for all human neutrophil α-defensins.

Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants.

BACKGROUND: HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. METHODS: We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. RESULTS: By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. CONCLUSIONS: The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. GENERAL SIGNIFICANCE: Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.

Self-Assembling Myristoylated Human α-Defensin 5 as a Next-Generation Nanobiotics Potentiates Therapeutic Efficacy in Bacterial Infection.

The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human α-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli ( E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.

Human Enteric α-Defensin 5 Promotes Shigella Infection by Enhancing Bacterial Adhesion and Invasion.

Shigella is a Gram-negative bacterium that causes bacillary dysentery worldwide. It invades the intestinal epithelium to elicit intense inflammation and tissue damage, yet the underlying mechanisms of its host selectivity and low infectious inoculum remain perplexing. Here, we report that Shigella co-opts human α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, to enhance its adhesion to and invasion of mucosal tissues. HD5 promoted Shigella infection in vitro in a structure-dependent manner. Shigella, commonly devoid of an effective host-adhesion apparatus, preferentially targeted HD5 to augment its ability to colonize the intestinal epithelium through interactions with multiple bacterial membrane proteins. HD5 exacerbated infectivity and Shigella-induced pathology in a culture of human colorectal tissues and three animal models. Our findings illuminate how Shigella exploits innate immunity by turning HD5 into a virulence factor for infection, unveiling a mechanism of action for this highly proficient human pathogen.

The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses Neuroblastoma Growth and Metastasis.

Soluble pulmonary factors have been reported to be capable of inhibiting the viability of cancer cells that metastasize to the lung, but the molecular identity was obscure. Here we report the isolation and characterization of the beta subunit of hemoglobin as a lung-derived antimetastatic factor. Peptide mapping in the beta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. In tissue culture, both HBB and murine HBB2 mediated growth arrest and apoptosis of lung-metastasizing neuroblastoma cells, along with a variety of other human cancer cell lines. Metox acted similarly and its administration in human tumor xenograft models limited the development of adrenal neuroblastoma tumors as well as spontaneous lung and bone marrow metastases. Expression studies in mice indicated that HBB2 is produced by alveolar epithelial and endothelial cells and is upregulated in mice bearing undetectable metastasis. Our work suggested a novel function for HBB as a theranostic molecule: an innate antimetastasis factor with potential utility as an anticancer drug and a biomarker signaling the presence of clinically undetectable metastasis. Cancer Res; 77(1); 14-26. ©2016 AACR.

Activity of Genital Tract Secretions and Synthetic Antimicrobial Peptides against Group B Streptococcus.

PROBLEM: Genital tract secretions inhibit Escherichia coli (E. coli) through antimicrobial peptides (AMP) secreted by the host and vaginal microbiota. However, there are limited data against group B Streptococcus (GBS). METHOD OF STUDY: Group B Streptococcus were incubated with cervico-vaginal lavage (CVL) samples from healthy non-pregnant women (n = 12) or synthetic AMP and monitored for bacterial growth using a turbidimetric approach. E. coli inhibitory activity was determined by a colony-forming unit assay. RESULTS: None of the CVL samples inhibited GBS. The human neutrophil peptide-1 and human defensin 5 inhibited GBS growth by ≥80% at concentrations ≥20 µg/mL and ≥50 µg/mL, respectively, while human beta-defensin 2 and LL-37 did not inhibit at highest concentration tested (100 µg/mL). In contrast, all AMP inhibited E. coli. CONCLUSIONS: Antimicrobial peptides may protect against E. coli colonization but have more limited activity against GBS. Future studies will focus on augmenting host defense with specific AMP to prevent genitourinary infection with these pathogenic organisms.

Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function.

HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr¹6, Ile²°, Leu²5, and Phe²8. Previously, alanine scanning mutagenesis identified each of these residues and other hydrophobic residues as important for function. Here we report further structural and functional studies of residues shown to interact with one another across oligomeric interfaces: I20A-HNP1 and L25A-HNP1, plus the double alanine mutants I20A/L25A-HNP1 and Y16A/F28A-HNP1, and the quadruple alanine mutant Y16A/I20A/L25A/F28A-HNP1. We tested binding to HIV-1 gp120 and HNP1 by surface plasmon resonance, binding to HIV-1 gp41 and HNP1 by fluorescence polarization, inhibition of anthrax lethal factor, and antibacterial activity using the virtual colony count assay. Similar to the previously described single mutant W26A-HNP1, the quadruple mutant displayed the least activity in all functional assays, followed by the double mutant Y16A/F28A-HNP1. The effects of the L25A and I20A single mutations were milder than the double mutant I20A/L25A-HNP1. Crystallographic studies confirmed the correct folding and disulfide pairing, and depicted an array of dimeric and tetrameric structures. These results indicate that side chain hydrophobicity is the critical factor that determines activity at these positions.

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions.

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G(o)) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.

The metastatic microenvironment: lung-derived factors control the viability of neuroblastoma lung metastasis.

Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations-lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung-derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro-apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung-derived factors affected various additional progression-linked cellular characteristics of neuroblastoma cells, such as the expression of stem-cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease.

Structural and functional analysis of the pro-domain of human cathelicidin, LL-37.

Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.

An ultrahigh affinity d-peptide antagonist Of MDM2.

The oncoprotein MDM2 negatively regulates the activity and stability of the p53 tumor suppressor and is an important molecular target for anticancer therapy. Aided by mirror image phage display and native chemical ligation, we have previously discovered several proteolysis-resistant duodecimal d-peptide antagonists of MDM2, termed (D)PMI-α, ß, γ. The prototypic d-peptide inhibitor (D)PMI-α binds ((25-109))MDM2 at an affinity of 220 nM and kills tumor cells in vitro and inhibits tumor growth in vivo by reactivating the p53 pathway. Herein, we report the design of a superactive d-peptide antagonist of MDM2, termed (D)PMI-δ, of which the binding affinity for ((25-109))MDM2 has been improved over (D)PMI-α by 3 orders of magnitude (K(d) = 220 pM). X-ray crystallographic studies validate (D)PMI-δ as an exceedingly potent inhibitor of the p53-MDM2 interaction, promising to be a highly attractive lead drug candidate for anticancer therapeutic development.