Peptide Stability Is Important but Not a General Requirement for Antimicrobial and Antibiofilm Activity In Vitro and In Vivo.

Peptide stability to proteases has been a major requirement for developing peptide therapeutics. This study investigates the effects of peptide stability on antimicrobial and antibiofilm activity under various conditions. For this purpose, two human cathelicidin-derived peptides differing in stability to proteases were utilized. While GF-17, a peptide derived from the major antimicrobial region of human LL-37, can be rapidly cleaved by proteases, the engineered peptide 17BIPHE2 is resistant to multiple proteases. In the standard antimicrobial susceptibility, killing kinetics, and membrane permeabilization assays conducted in vitro using planktonic bacteria, these two peptides displayed similar potency. The two peptides were also similarly active against methicillin-resistant Staphylococcus aureus (MRSA) USA300 prior to biofilm formation. However, 17BIPHE2 was superior to GF-17 in disrupting preformed biofilms probably due to both enhanced stability and slightly higher DNA binding capacity. In a wax moth model, 17BIPHE2 better protected insects from MRSA infection-caused death than GF-17, consistent with the slower degradation of 17BIPHE2 than GF-17. Here, peptide antimicrobial activity was found to be critical for in vivo efficacy. When incorporated in the nanofiber/microneedle delivery device, GF-17 and 17BIPHE2 displayed a similar effect in eliminating MRSA in murine chronic wounds, underscoring the advantage of nanofibers in protecting the peptide from degradation. Since nanoformulation can ease the requirement of peptide stability, it opens the door to a direct use of natural peptides or their cocktails for antimicrobial treatment, accelerating the search of effective antibiofilm peptides to treat chronic wounds.

Two distinct amphipathic peptide antibiotics with systemic efficacy.

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.

Low cationicity is important for systemic in vivo efficacy of database-derived peptides against drug-resistant Gram-positive pathogens.

As bacterial resistance to traditional antibiotics continues to emerge, new alternatives are urgently needed. Antimicrobial peptides (AMPs) are important candidates. However, how AMPs are designed with in vivo efficacy is poorly understood. Our study was designed to understand structural moieties of cationic peptides that would lead to their successful use as antibacterial agents. In contrast to the common perception, serum binding and peptide stability were not the major reasons for in vivo failure in our studies. Rather, our systematic study of a series of peptides with varying lysines revealed the significance of low cationicity for systemic in vivo efficacy against Gram-positive pathogens. We propose that peptides with biased amino acid compositions are not favored to associate with multiple host factors and are more likely to show in vivo efficacy. Thus, our results uncover a useful design strategy for developing potent peptides against multidrug-resistant pathogens.

Design of Antimicrobial Peptides: Progress Made with Human Cathelicidin LL-37.

The incorporation of the innate immune system into humans is essential for survival and health due to the rapid replication of invading microbes and the delayed action of the adaptive immune system. Antimicrobial peptides are important components of human innate immunity. Over 100 such peptides have been identified in various human tissues. Human cathelicidin LL-37 is best studied, and there has been a growing interest in designing new peptides based on LL-37. This chapter describes the alternative processing of the human cathelicidin precursor, protease digestion, and lab cutting of LL-37. Both a synthetic peptide library and structure-based design are utilized to identify the active regions. Although challenging, the determination of the 3D structure of LL-37 enabled the identification of the core antimicrobial region. The minimal region of LL-37 can be function-dependent. We discuss the design and potential applications of LL-37 into antibacterial, antibiofilm, antiviral, antifungal, immune modulating, and anticancer peptides. LL-37 has been engineered into 17BIPHE2, a stable, selective, and potent antimicrobial, antibiofilm, and anticancer peptide. Both 17BIPHE2 and SAAP-148 can eliminate the ESKAPE pathogens and show topical in vivo antibiofilm efficacy. Also discussed are other application strategies, including peptide formulation, antimicrobial implants, and peptide-inducing factors such as vitamin D and sunlight. Finally, we summarize what we learned from peptide design based on human LL-37.

The π Configuration of the WWW Motif of a Short Trp-Rich Peptide Is Critical for Targeting Bacterial Membranes, Disrupting Preformed Biofilms, and Killing Methicillin-Resistant Staphylococcus aureus.

Tryptophan-rich peptides, being short and suitable for large-scale chemical synthesis, are attractive candidates for developing a new generation of antimicrobials to combat antibiotic-resistant bacteria (superbugs). Although there are numerous pictures of the membrane-bound structure of a single tryptophan (W), how multiple Trp amino acids assemble themselves and interact with bacterial membranes is poorly understood. This communication presents the three-dimensional structure of an eight-residue Trp-rich peptide (WWWLRKIW-NH2 with 50% W) determined by the improved two-dimensional nuclear magnetic resonance method, which includes the measurements of 13C and 15N chemical shifts at natural abundance. This peptide forms the shortest two-turn helix with a distinct amphipathic feature. A unique structural arrangement is identified for the Trp triplet, WWW, that forms a π configuration with W2 as the horizontal bar and W1/W3 forming the two legs. An arginine scan reveals that the WWW motif is essential for killing methicillin-resistant Staphylococcus aureus USA300 and disrupting preformed bacterial biofilms. This unique π configuration for the WWW motif is stabilized by aromatic-aromatic interactions as evidenced by ring current shifts as well as nuclear Overhauser effects. Because the WWW motif is maintained, a change of I7 to R led to a potent antimicrobial and antibiofilm peptide with 4-fold improvement in cell selectivity. Collectively, this study elucidated the structural basis of antibiofilm activity of the peptide, identified a better peptide candidate via structure-activity relationship studies, and laid the foundation for engineering future antibiotics based on the WWW motif.

Arginine-lysine positional swap of the LL-37 peptides reveals evolutional advantages of the native sequence and leads to bacterial probes.

Antimicrobial peptides are essential components of the innate immune system of multicellular organisms. Although cationic and hydrophobic amino acids are known determinants of these amphipathic molecules for bacterial killing, it is not clear how lysine-arginine (K-R) positional swaps influence peptide structure and activity. This study addresses this question by investigating two groups of peptides (GF-17 and 17BIPHE2) derived from human cathelicidin LL-37. K-R positional swap showed little effect on minimal inhibitory concentrations of the peptides. However, there are clear differences in bacterial killing kinetics. The membrane permeation patterns vary with peptide and bacterial types, but not changes in fluorescent dyes, salts or pH. In general, the original peptide is more efficient in bacterial killing, but less toxic to human cells, than the K-R swapped peptides, revealing the evolutionary significance of the native sequence for host defense. The characteristic membrane permeation patterns for different bacteria suggest a possible application of these K-R positional-swapped peptides as molecular probes for the type of bacteria. Such differences are related to bacterial membrane compositions: minimal for Gram-positive Staphylococcus aureus with essentially all anionic lipids (cardiolipin and phosphatidylglycerol), but evident for Gram-negative Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli with a mixture of phosphatidylethanolamine and phosphatidylglycerol. Biophysical characterization found similar structures and binding affinities for these peptides in vesicle systems mimicking E. coli and S. aureus. It seems that interfacial arginines of GF-17 are preferred over lysines in bacterial membrane permeation. Our study sheds new light on the design of cationic amphipathic peptides.

Small molecule mimics of DFTamP1, a database designed anti-Staphylococcal peptide.

Antimicrobial peptides (AMPs) are important templates for developing new antimicrobial agents. Previously, we developed a database filtering technology that enabled us to design a potent anti-Staphylococcal peptide DFTamP1. Using this same design approach, we now report the discovery of a new class of bis-indole diimidazolines as AMP small molecule mimics. The best compound killed multiple S. aureus clinical strains in both planktonic and biofilm forms. The compound appeared to target bacterial membranes with antimicrobial activity and membrane permeation ability similar to daptomycin.

Decoding the functional roles of cationic side chains of the major antimicrobial region of human cathelicidin LL-37.

Human cathelicidin LL-37 is a critical cationic antimicrobial peptide for host defense against infection, immune modulation, and wound healing. This article elucidates the functional roles of the cationic side chains of the major antimicrobial region of LL-37, corresponding to residues 17 to 32 (designated GF-17). Antimicrobial assays, killing kinetics studies, and vesicle leakage experiments all indicate that a conversion of lysines to arginines affected the ability of the peptide to kill the Gram-positive Staphylococcus aureus strain USA300. Alanine scanning experiments show that S. aureus is less sensitive than Escherichia coli to a single cationic residue mutation of GF-17. Among the five cationic residues, R23 appears to be somewhat important in killing S. aureus. However, R23 and K25 of GF-17 are of prime importance in killing the Gram-negative organism E. coli. In particular, R23 is essential for (i) rapid recognition, (ii) permeation of the E. coli outer membrane, (iii) clustering of anionic lipids in a membrane system mimicking the E. coli inner membrane, and (iv) membrane disruption. Bacterial aggregation (i.e., rapid recognition via charge neutralization) is the first step of the peptide action. Structurally, R23 is located in the interface (i.e., the first action layer), a situation ideal for the interactions listed above. In contrast, residues K18, R19, and R29 are on the hydrophilic surface of the amphipathic helix and play only a secondary role. Mapping of the functional spectrum of cationic residues of GF-17 provides a solid basis for engineering bacterium-specific antimicrobials using this highly potent template.

Short and Robust Anti-Infective Lipopeptides Engineered Based on the Minimal Antimicrobial Peptide KR12 of Human LL-37.

This study aims to push the frontier of the engineering of human cathelicidin LL-37, a critical antimicrobial innate immune peptide that wards off invading pathogens. By sequential truncation of the smallest antibacterial peptide (KR12) of LL-37 and conjugation with fatty acids, with varying chain lengths, a library of lipopeptides is generated. These peptides are subjected to antibacterial activity and hemolytic assays. Candidates (including both forms made of l- and d-amino acids) with the optimal cell selectivity are subsequently fed to the second layer of in vitro filters, including salts, pH, serum, and media. These practices lead to the identification of a miniature LL-37 like peptide (d-form) with selectivity, stability, and robust antimicrobial activity in vitro against both Gram-positive and negative bacteria. Proteomic studies reveal far fewer serum proteins that bind to the d-form than the l-form peptide. C10-KR8d targets bacterial membranes to become helical, making it difficult for bacteria to develop resistance in a multiple passage experiment. In vivo, C10-KR8d is able to reduce bacterial burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 LAC in neutropenic mice. In addition, this designer peptide prevents bacterial biofilm formation in a catheter-associated mouse model. Meanwhile, C10-KR8d also recruits cytokines to the vicinity of catheters to clear infection. Thus, based on the antimicrobial region of LL-37, this study succeeds in identifying the smallest anti-infective peptide C10-KR8d with both robust antimicrobial, antibiofilm, and immune modulation activities.

Sequence Permutation Generates Peptides with Different Antimicrobial and Antibiofilm Activities.

Antibiotic resistance poses a threat to our society, and 10 million people could die by 2050. To design potent antimicrobials, we made use of the antimicrobial peptide database (APD). Using the database filtering technology, we identified a useful template and converted it into an effective peptide WW291 against methicillin-resistant Staphylococcus aureus (MRSA). Here, we compared the antibacterial activity and cytotoxicity of a family of peptides obtained from sequence permutation of WW291. The resulting eight WW peptides (WW291-WW298) gained different activities against a panel of bacteria. While WW295 inhibited the growth of Escherichia coli, WW298 was highly active against S. aureus USA300 LAC. Consistently with this, WW298 was more effective in permeating or depolarizing the S. aureus membranes, whereas WW295 potently permeated the E. coli membranes. In addition, WW298, but not WW295, inhibited the MRSA attachment and could disrupt its preformed biofilms more effectively than daptomycin. WW298 also protected wax moths Galleria mellonella from MRSA infection causing death. Thus, sequence permutation provides one useful avenue to generating antimicrobial peptides with varying activity spectra. Taken together with amino acid composition modulation, these methods may lead to narrow-spectrum peptides that are more promising to selectively eliminate invading pathogens without damaging commensal microbiota.

Modulation of antimicrobial potency of human cathelicidin peptides against the ESKAPE pathogens and in vivo efficacy in a murine catheter-associated biofilm model.

Antimicrobial peptides are essential components of innate immune systems that protect hosts from infection. They are also useful candidates for developing a new generation of antibiotics to fight antibiotic-resistant pathogens. Human innate immune peptide LL-37 can inhibit biofilm formation, but suffers from high cost due to a long peptide length and rapid protease degradation. To improve the peptide, we previously identified the major active region and changed the peptide backbone structure. This study designed two families of new peptides by altering peptide side chains. Interestingly, these peptides displayed differential potency against various ESKAPE pathogens in vitro and substantially reduced hemolysis. Further potency test in vivo revealed that 17tF-W eliminated the burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 in both mouse-embedded catheters and their surrounding tissues. In addition, peptide treatment suppressed the level of chemokine TNFα, and boosted the levels of chemokines MCP-1, IL-17A and IL-10 in the surrounding tissues of the infected catheter embedded in mice. In conclusion, we have designed a set of new LL-37 peptides with varying antimicrobial activities, opening the door to potential topical treatment of infections involving different drug-resistant pathogens.

Amino Acid Composition Determines Peptide Activity Spectrum and Hot-Spot-Based Design of Merecidin.

There is a great interest in developing the only human cathelicidin into therapeutic molecules. The major antimicrobial region of human LL-37 corresponds to residues 17-32. The resultant peptide GF-17 shows a broad spectrum of antimicrobial activity against both Gram-positive and negative bacteria. By reducing the hydrophobic content, we previously succeeded in converting the broad-spectrum GF-17 to two narrow-spectrum peptides (GF-17d3 and KR-12) with activity against Gram-negative bacteria. This study demonstrates that substitution of multiple basic amino acids by hydrophobic alanines makes a broad-spectrum peptide 17BIPHE2 (designed based on GF-17d3) active against Staphylococcal pathogens but not other bacteria tested. Taken together, our results reveal distinct charge and hydrophobic requirements for peptides to kill Gram-positive or Gram-negative bacteria. This finding is in line with the bioinformatics analysis of the peptides in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP). In addition, a hot spot arginine is identified and used to design merecidin with reduced toxicity to human cells. Merecidin protects wax moth larvae (Galleria mellonella) from the infection of methicillin-resistant S. aureus USA300. These new selective peptides constitute interesting candidates for future development.

Antibacterial, antifungal, anticancer activities and structural bioinformatics analysis of six naturally occurring temporins.

Antimicrobial peptides are a special class of natural products with potential applications as novel therapeutics. This study focuses on six temporins (four with no activity data and two as positive controls). Using synthetic peptides, we report antibacterial, antifungal, and anticancer activities of temporins-CPa, CPb, 1Ga, 1Oc, 1Ola, and 1SPa. While temporin-1Ga and temporin-1OLa showed higher antifungal and anticancer activity, most of these peptides were active primarily against Gram-positive bacteria. Temporin-1OLa, with the highest cell selectivity index, could preferentially kill methicillin-resistant Staphylococcus aureus (MRSA), consistent with a reduced hemolysis in the presence of bacteria. Mechanistically, temporin-1OLa rapidly killed MRSA by damaging bacterial membranes. Using micelles as a membrane-mimetic model, we determined the three-dimensional structure of temporin-1OLa by NMR spectroscopy. The peptide adopted a two-domain structure where a hydrophobic patch is followed by a classic amphipathic helix covering residues P3-I12. Such a structure is responsible for anti-biofilm ability in vitro and in vivo protection of wax moths Galleria mellonella from staphylococcal infection. Finally, our bioinformatic analysis leads to a classification of temporins into six types and confers significance to this NMR structure since temporin-1OLa shares a sequence model with 62% of temporins. Collectively, our results indicate the potential of temporin-1OLa as a new anti-MRSA compound, which shows an even better anti-biofilm capability in combination with linezolid.

Design and surface immobilization of short anti-biofilm peptides.

Short antimicrobial peptides are essential to keep us healthy and their lasting potency can inspire the design of new types of antibiotics. This study reports the design of a family of eight-residue tryptophan-rich peptides (TetraF2W) obtained by converting the four phenylalanines in temporin-SHf to tryptophans. The temporin-SHf template was identified from the antimicrobial peptide database (http://aps.unmc.edu/AP). Remarkably, the double arginine variant (TetraF2W-RR) was more effective in killing methicillin-resistant Staphylococcus aureus (MRSA) USA300, but less cytotoxic to human skin HaCat and kidney HEK293 cells, than the lysine-containing dibasic combinations (KR, RK and KK). Killing kinetics and fluorescence spectroscopy suggest membrane targeting of TetraF2W-RR, making it more difficult for bacteria to develop resistance. Because established biofilms on medical devices are difficult to remove, we chose to covalently immobilize TetraF2W-RR onto the polyethylene terephthalate (PET) surface to prevent biofilm formation. The successful surface coating of the peptide is supported by FT-IR and XPS spectroscopies, chemical quantification, and antibacterial assays. This peptide-coated surface indeed prevented S. aureus biofilm formation with no cytotoxicity to human cells. In conclusion, TetraF2W-RR is a short Trp-rich peptide with demonstrated antimicrobial and anti-biofilm potency against MRSA in both the free and immobilized forms. Because these short peptides can be synthesized cost effectively, they may be developed into new antimicrobial agents or used as surface coating compounds. STATEMENT OF SIGNIFICANCE: It is stunning that the total deaths due to methicillin-resistant Staphylococcus aureus (MRSA) infection are comparable to AIDS/HIV-1, making it urgent to explore new possibilities. This study deals with this problem by two strategies. First, we have designed a family of novel antimicrobial peptides with merely eight amino acids, making it cost effective for chemical synthesis. These peptides are potent against MRSA USA300. Our study uncovers that the high potency of the tryptophan-rich short peptide is coupled with arginines, whereas these Trp- and Arg-rich peptides are less toxic to select human cells than the lysine-containing analogs. Such a combination generates a more selective peptide. As a second strategy, we also demonstrate successful covalent immobilization of this short peptide to the polyethylene terephthalate (PET) surface by first using a chitosan linker, which is easy to obtain. Because biofilms on medical devices are difficult to remove by traditional antibiotics, we also show that the peptide coated surface can prevent biofilm formation. Although rarely demonstrated, we provide evidence that both the free and immobilized peptides target bacterial membranes, rendering it difficult for bacteria to develop resistance. Collectively, the significance of our study is the design of novel antimicrobial peptides provides a useful template for developing novel antimicrobials against MRSA. In addition, orientation-specific immobilization of the same short peptide can prevent biofilm formation on the PET surface, which is widely used in making prosthetic heart valves cuffs and other bio devices.

Anti-Staphylococcal Biofilm Effects of Human Cathelicidin Peptides.

Staphylococcus aureus can live together in the form of biofilms to avoid elimination by the host. Thus, a useful strategy to counteract bacterial biofilms is to re-engineer human antimicrobial peptide LL-37 so that it can be used as a remedy for preventing and removing biofilms. This study reports antibiofilm effects of four human cathelicidin LL-37 peptides against community-associated and hospital isolated methicillin-resistant Staphylococcus aureus (MRSA) strains. Although the intact molecule LL-37 inhibited biofilm formation at low concentrations, it did not inhibit bacterial attachment nor disrupt preformed biofilms. However, two 17-residue peptides, GF-17 and 17BIPHE2, inhibited bacterial attachment, biofilm growth, and disrupted established biofilms. An inactive peptide RI-10 was used as a negative control. Our results obtained using the S. aureus mutants in a static biofilm model are consistent with the literature obtained in a flow cell biofilm model. Because 17BIPHE2 is the most effective biofilm disruptor with desired stability to proteases, it is a promising lead for developing new anti-MRSA biofilm agents.

Small lipopeptides possess anti-biofilm capability comparable to daptomycin and vancomycin.

Antibiotic resistance, to a large extent, is related to the formation of bacterial biofilms. Thus, compounds with anti-biofilm capability are of practical importance. Inspired by the recent discovery of two amino acid lipopeptides from marine bacteria, we constructed a family of small lipopeptides with 2-3 amino acids. While no antimicrobial activity was found for anionic lipopeptides, cationic candidates are potent against Staphylococcus strains, such as methicillin-resistant Staphylococcus aureus (MRSA) USA200, USA300, USA400, UAMS-1, Newman, and Mu50. In the simplest design, two lysines (C14-KK) or three arginines (C14-RRR) attached to an acyl chain of 14 carbons were sufficient to make the compounds antimicrobial. These simple lipopeptides are inherently stable towards S. aureus V8 proteinase and fungal proteinase K, more soluble in water, and more selective than other lipopeptides containing a mixture of hydrophobic and cationic amino acids. Furthermore, the activity of C14-RRR was not compromised by salts, serum, or a change in pH. Live cell experiments revealed that these lipopeptides, with a detergent-like structure, killed bacteria rapidly by targeting cell membranes. Importantly, these compounds were also able to inhibit biofilm formation and could even disrupt preformed biofilms of clinically relevant MRSA strains with an in vitro efficacy comparable to daptomycin and vancomycin. These results indicate that small lipopeptides are potentially useful candidates for preventing or eliminating bacterial biofilms alone or in combination with daptomycin or vancomycin.

Antimicrobial peptides in 2014.

This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

Transformation of human cathelicidin LL-37 into selective, stable, and potent antimicrobial compounds.

This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, the peptide (17BIPHE2) gained activity against the ESKAPE pathogens, including Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. In vitro, 17BIPHE2 could disrupt bacterial membranes and bind to DNA. In vivo, the peptide prevented staphylococcal biofilm formation in a mouse model of catheter-associated infection. Meanwhile, it boosted the innate immune response to further combat the infection. Because these peptides are potent, cell-selective, and stable to several proteases, they may be utilized to combat one or more ESKAPE pathogens.

Mdm2 promotes genetic instability and transformation independent of p53.

Mdm2, a regulator of the tumor suppressor p53, is frequently overexpressed in human malignancies. Mdm2 also has unresolved, p53-independent functions that contribute to tumorigenesis. Here, we show that increased Mdm2 expression induced chromosome/chromatid breaks and delayed DNA double-strand break repair in cells lacking p53 but not in cells with a mutant form of Nbs1, a component of the Mre11/Rad50/Nbs1 DNA repair complex. A 31-amino-acid region of Mdm2 was necessary for binding to Nbs1. Mutation of conserved amino acids in the Nbs1 binding domain of Mdm2 inhibited Mdm2-Nbs1 association and prevented Mdm2 from delaying phosphorylation of H2AX and ATM-S/TQ sites, repair of DNA breaks, and resolution of DNA damage foci. Similarly, the mutation of eight amino acids in the Mdm2 binding domain of Nbs1 inhibited Mdm2-Nbs1 interaction and blocked the ability of Mdm2 to delay DNA break repair. Both Nbs1 and ATM, but not the ubiquitin ligase activity of Mdm2, were necessary to inhibit DNA break repair. Only Mdm2 with an intact Nbs1 binding domain was able to increase the frequency of chromosome/chromatid breaks and the transformation efficiency of cells lacking p53. Therefore, the interaction of Mdm2 with Nbs1 inhibited DNA break repair, leading to chromosome instability and subsequent transformation that was independent of p53.

A novel nuclear interactor of ARF and MDM2 (NIAM) that maintains chromosomal stability.

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.