Search

Mutant KRAS is a major driver of oncogenesis in a multitude of cancers but remains a challenging target for classical small molecule drugs, motivating the exploration of alternative approaches. Here, we show that aggregation-prone regions (APRs) in the primary sequence of the oncoprotein constitute intrinsic vulnerabilities that can be exploited to misfold KRAS into protein aggregates. Conveniently, this propensity that is present in wild-type KRAS is increased in the common oncogenic mutations at positions 12 and 13. We show that synthetic peptides (Pept-ins™) derived from two distinct KRAS APRs could induce the misfolding and subsequent loss of function of oncogenic KRAS, both of recombinantly produced protein in solution, during cell-free translation and in cancer cells. The Pept-ins exerted antiproliferative activity against a range of mutant KRAS cell lines and abrogated tumor growth in a syngeneic lung adenocarcinoma mouse model driven by mutant KRAS G12V. These findings provide proof-of-concept that the intrinsic misfolding propensity of the KRAS oncoprotein can be exploited to cause its functional inactivation.

Amyloid-like aggregation of proteins is induced by short amyloidogenic sequence segments within a specific protein sequence resulting in self-assembly into β-sheets. We recently validated a technology platform in which synthetic amyloid peptides ("Pept-ins") containing a specific aggregation-prone region (APR) are used to induce specific functional knockdown of the target protein from which the APR was derived, including bacterial, viral, and mammalian cell proteins. In this work, we investigated if Pept-ins can be used as vector probes for in vivo Positron Emission Tomography (PET) imaging of intracellular targets. The radiolabeled Pept-ins [68Ga]Ga-NODAGA-PEG4-vascin (targeting VEGFR2) and [68Ga]Ga-NODAGA-PEG2-P2 (targeting E. coli) were evaluated as PET probes. The Pept-in based radiotracers were cross-validated in a murine tumor and muscle infection model, respectively, and were found to combine target specificity with favorable in vivo pharmacokinetics. When the amyloidogenicity of the interacting region of the peptide is suppressed by mutation, cellular uptake and in vivo accumulation are abolished, highlighting the importance of the specific design of synthetic Pept-ins. The ubiquity of target-specific amyloidogenic sequence segments in natural proteins, the straightforward sequence-based design of the Pept-in probes, and their spontaneous internalization by cells suggest that Pept-ins may constitute a generic platform for in vivo PET imaging of intracellular targets. ispartof: BIOCONJUGATE CHEMISTRY vol:32 issue:9 pages:2052-2064 ispartof: location:United States status: published

Amyloid-like aggregation of proteins is induced by short amyloidogenic sequence segments within a specific protein sequence resulting in self-assembly into β-sheets. We recently validated a technology platform in which synthetic amyloid peptides ("Pept-ins") containing a specific aggregation-prone region (APR) are used to induce specific functional knockdown of the target protein from which the APR was derived, including bacterial, viral, and mammalian cell proteins. In this work, we investigated if Pept-ins can be used as vector probes for

Despite our extensive knowledge of the genetic regulation of heat shock proteins (HSPs), the evolutionary routes that allow bacteria to adaptively tune their HSP levels and corresponding proteostatic robustness have been explored less. In this report, directed evolution experiments using the Escherichia coli model system unexpectedly revealed that seemingly random single mutations in its

Decades of research into bacterial persistence has been unable to fully characterize this antibiotic-tolerant phenotype, thereby hampering the development of therapies effective against chronic infections. Although some active persister mechanisms have been identified, the prevailing view is that cells become persistent because they enter a dormant state. We therefore characterized starvation-induced dormancy in Escherichia coli. Our findings indicate that dormancy develops gradually; persistence strongly increases during stationary phase and decreases again as persisters enter the viable but nonculturable (VBNC) state. Importantly, we show that dormancy development is tightly associated with progressive protein aggregation, which occurs concomitantly with ATP depletion during starvation. Persisters contain protein aggregates in an early developmental stage, while VBNC cells carry more mature aggregates. Finally, we show that at least one persister protein, ObgE, works by triggering aggregation, even at endogenous levels, and thereby changing the dynamics of persistence and dormancy development. These findings provide evidence for a genetically controlled, gradual development of persisters and VBNC cells through protein aggregation. IMPORTANCE While persistence and the viable but nonculturable (VBNC) state are currently investigated in isolation, our results strongly indicate that these phenotypes represent different stages of the same dormancy program and that they should therefore be studied within the same conceptual framework. Moreover, we show here for the first time that the dynamics of protein aggregation perfectly match the onset and further development of bacterial dormancy and that different dormant phenotypes are linked to different stages of protein aggregation. Our results thereby strongly hint at a causal relationship between both. Because many conditions known to trigger persistence are also known to influence aggregation, it is tempting to speculate that a variety of different persister pathways converge at the level of protein aggregation. If so, aggregation could emerge as a general principle that underlies the development of persistence which could be exploited for the design of antipersister therapies. ispartof: MBIO vol:12 issue:4 ispartof: location:United States status: published

Aggregation can be selectively induced by aggregation-prone regions (APRs) contained in the target proteins. Aggregation-inducing antimicrobial peptides (Pept-ins) contain sequences homologous to APRs of target proteins and exert their bactericidal effect by causing aggregation of a large number of proteins. To better understand the mechanism of action of Pept-ins and the resistance mechanisms, we analyzed the phenotypic, lipidomic, and transcriptomic as well as genotypic changes in laboratory-derived Pept-in-resistant E. coli mutator cells. The analysis showed that the Pept-in resistance mechanism is dominated by a decreased Pept-in uptake, in both laboratory-derived mutator cells and clinical isolates. Our data indicate that Pept-in uptake involves an electrostatic attraction between the Pept-in and the bacterial membrane and follows a complex mechanism potentially involving many transporters. Furthermore, it seems more challenging for bacteria to become resistant toward Pept-ins that are less dependent on electrostatic attraction for uptake, suggesting that future Pept-ins should be selected for this property. ispartof: CELL CHEMICAL BIOLOGY vol:28 issue:4 pages:524-+ ispartof: location:United States status: published

Decades of research into bacterial persistence has been unable to fully characterize this antibiotic-tolerant phenotype, thereby hampering the development of therapies effective against chronic infections. Although some active persister mechanisms have been identified, the prevailing view is that cells become persistent because they enter a dormant state. We therefore characterized starvation-induced dormancy in Escherichia coli. Our findings indicate that dormancy develops gradually; persistence strongly increases during stationary phase and decreases again as persisters enter the viable but nonculturable (VBNC) state. Importantly, we show that dormancy development is tightly associated with progressive protein aggregation, which occurs concomitantly with ATP depletion during starvation. Persisters contain protein aggregates in an early developmental stage while VBNC cells carry more mature aggregates. Finally, we show that at least one persister protein, ObgE, works by triggering aggregation and thereby changing the dynamics of persistence and dormancy development. These findings provide evidence for a genetically-controlled, gradual development of persisters and VBNC cells through protein aggregation.

Summary Objectives The opportunistic pathogen Staphylococcus epidermidis is progressively involved in device-related infections. Since these infections involve biofilm formation, antibiotics are not effective. Conversely, a vaccine can be advantageous to prevent these infections. In view of vaccine development, predicted surface proteins were evaluated on their potential as a vaccine target. Methods Immunoglobulins directed against S. epidermidis surface proteins SesB, M, O, Q and R were used to firstly affirm their surface location. Further, inhibitory effects of these IgGs on biofilm formation were determined in vitro on polystyrene and polyurethane surfaces and in vivo using a subcutaneous catheter mouse model. We also examined the opsonophagocytotic capacity of these IgGs. Results Surface localization of the five Ses proteins was demonstrated both for planktonic and sessile cells, though to a variable extent. Ses-specific IgGs added to planktonic cells had a variable inhibitory effect on cell adhesion to polystyrene, while only anti-SesO IgGs decreased cell attachment to polyurethane catheters. Although phagocytic killing was only obtained after opsonization with SesB-specific IgGs, a significant reduction of in vivo formed biofilms was observed after administration of SesB-, SesM- and SesO-specific IgGs. Conclusions Regardless of their characterization or function, S. epidermidis surface proteins can be adequate targets for vaccine development aiming the prevention of device-related infections caused by invasive S. epidermidis strains.

There is evidence that pathogenic bacteria can adapt to antiseptics upon repeated exposure. More alarming is the concomitant increase in antibiotic resistance that has been described for some pathogens. Unfortunately, effects of adaptation and cross-adaptation are hardly known for oral pathogens, which are very frequently exposed to antiseptics. Therefore, this study aimed to determine the in vitro increase in minimum inhibitory concentrations (MICs) in oral pathogens after repeated exposure to chlorhexidine or cetylpyridinium chloride, to examine if (cross-)adaptation to antiseptics/antibiotics occurs, if (cross-)adaptation is reversible and what the potential underlying mechanisms are. When the pathogens were exposed to antiseptics, their MICs significantly increased. This increase was in general at least partially conserved after regrowth without antiseptics. Some of the adapted species also showed cross-adaptation, as shown by increased MICs of antibiotics and the other antiseptic. In most antiseptic-adapted bacteria, cell-surface hydrophobicity was increased and mass-spectrometry analysis revealed changes in expression of proteins involved in a wide range of functional domains. These in vitro data shows the adaptation and cross-adaptation of oral pathogens to antiseptics and antibiotics. This was related to changes in cell surface hydrophobicity and in expression of proteins involved in membrane transport, virulence, oxidative stress protection and metabolism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish. ispartof: PLoS One vol:12 issue:7 ispartof: location:United States status: published

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

Aggregation by design Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model. Science , this issue p. 10.1126/science.aah4949

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325-4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325-4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325-4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325-4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325-4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate. ispartof: PLoS One vol:11 issue:1 ispartof: location:United States status: published

Staphylococcus epidermidis is one of the major concerns with respect to hospital-acquired infections. Therefore, a rapid and easy method to identify at species level S. epidermidis isolates out of a broad range of bacteria is necessary. Based on earlier studies, the sesC gene encoding a S. epidermidis surface protein revealed to be a highly conserved gene in this species. By means of an easy and inexpensive PCR assay, the presence of sesC was checked in 438 clinical staphylococcal isolates. Results showed that sesC is specifically present in all S. epidermidis. In conclusion, the sesC gene can be exploited as a genetic marker in order to distinguish S. epidermidis from other isolates.

Staphylococcus epidermidis account for the majority of foreign body-related infections particularly catheter-related infections. Its ability to adhere to materials and to promote formation of a biofilm is the most important feature of its pathogenicity. The presence of S. epidermidis surface components particularly SesC protein is essential for biofilm formation. Accordingly, in addition to antibiotic therapy has recently been a lot of attention on the development of vaccines against S. epidermidis surface proteins. The aim of this study is detection of biofilm phenotype of isolated S. epidermidis from respiratory catheters of hospitalized patients and evaluation of the effect of antibodies against SesC protein on biofilm formation. In this study, we've isolated 70 coagulase-negative staphylococcus strains from respiratory catheter samples (n=350). Then, 40 isolates of these strains were randomly selected; Twenty (50%) S. epidermidis strains were identified by Mass spectrometry from all 40 isolates. From these twenty strains, 30% produced dissolvable PIA-dependent biofilms in sodium metaperiodate after growth in Tryptic soy broth (TSB) with NaCl and 40% produced dissolvable protein-dependent biofilms in proteinase K after growth in TSB with glucose. Evaluated the effect of anti-SesC antibodies on biofilm formation by using a semi quantitative adherence assay. In 40% of cases, anti-SesC antibodies had significant inhibitory effect on biofilm formation (P

Human amyloids have been shown to interact with viruses and interfere with viral replication. Based on this observation, we employed a synthetic biology approach in which we engineered virus-specific amyloids against influenza A and Zika proteins. Each amyloid shares a homologous aggregation-prone fragment with a specific viral target protein. For influenza we demonstrate that a designer amyloid against PB2 accumulates in influenza A-infected tissue in vivo. Moreover, this amyloid acts specifically against influenza A and its common PB2 polymorphisms, but not influenza B, which lacks the homologous fragment. Our model amyloid demonstrates that the sequence specificity of amyloid interactions has the capacity to tune amyloid-virus interactions while allowing for the flexibility to maintain activity on evolutionary diverging variants., Some human amyloid proteins have been shown to interact with viral proteins, suggesting that they may have potential as therapeutic agents. Here the authors design synthetic amyloids specific for influenza A and Zika virus proteins, respectively, and show that they can inhibit viral replication.