Hydrophobic Amino Acid Substitutions Research Articles

Unlocking Antibacterial Potential: Key-Site-Based Regulation of Antibacterial Spectrum of Peptides.

In the pursuit of combating multidrug-resistant bacteria, antimicrobial peptides (AMPs) have emerged as promising agents; however, their application in clinical settings still presents challenges. Specifically, the exploration of crucial structural parameters that influence the antibacterial spectrum of AMPs and the subsequent development of tailored variants with either broad- or narrow-spectrum characteristics to address diverse clinical therapeutic needs has been overlooked. This study focused on investigating the effects of amino acid sites and hydrophobicity on the peptide's antibacterial spectrum through Ala scanning and fixed-point hydrophobic amino acid substitution techniques. The findings revealed that specific amino acid sites played a pivotal role in determining the antibacterial spectrum of AMPs and confirmed that broadening the spectrum could be achieved only by increasing hydrophobicity at certain positions. In conclusion, this research provided a theoretical basis for future precise regulation of an antimicrobial peptide's spectrum by emphasizing the intricate balance between amino acid sites and hydrophobicity.

Abstract 1851: Selective targeting of aberrant ETS transcription factors using uniquely modified mithramycin analogs

Abstract Background: Recent studies demonstrated that the aberrant oncogenic activity of ETS transcription factor fusions such as EWS-FLI1 and EWS-ERG in Ewing sarcoma can be targeted with the natural product mithramycin (MTM), which is known to have poor pharmacology. We hypothesized that the pharmacological properties of MTM can be ameliorated by rational semisynthetic modifications that improve its pharmacokinetic (PK) liabilities and expand its efficacy toxicity window. Further, we reasoned that the MTM core of each analog can facilitate DNA docking while the substitutions may interact uniquely with oncogenic ETS transcriptional complexes in different cancers. Here we present the pharmacologic properties of two analogs and their unique transcriptome profiles in TC32 Ewing sarcoma and VCaP prostate cancer cells, both of which depend on the aberrant oncogenic activity of an ETS gene fusion. Methods: MTM analogs were generated by semisynthetic conjugation to the 3-side chain of MTM. Cytotoxicity (GI50) was assessed using the resazurin assay following 72 hr incubation with test compounds (0.3nM-10uM). PK properties were assessed in athymic nu/nu mice after a bolus tail-vein injection and concentrations were assessed in plasma using LC/MS/MS methods. The maximum tolerated dose (MTD) was estimated based on weight loss and blood hematology. In vivo efficacy was assessed in subcutaneous xenografts established, which were dosed SIDx5 at the respective MTD or fractions of the MTD. Transcriptomic profiles were assessed in cells exposed to their respective 72 hr GI50 for 24 hr and in livers of mice that were dosed SIDx5. Results: Here we demonstrate that select modifications displayed unique cytotoxicity and efficacy Ewing sarcoma (EWS-FLI1) and VCaP prostate (TMPRSS2-ERG) cancer cell lines. Analogues with bulky amino acid substitutions in the 3-side chain of MTM had significantly improved PK in mice, as compared to MTM. Select analogs displayed selective efficacy in xenograft models of Ewing sarcoma at doses ranging as low as 1/3 of the MTD. Significantly, analogues of MTM displayed significantly different transcriptomic profiles in each cell line, which suggests that these molecules have unique properties despite sharing a core DNA-binding structure with MTM. Conclusions: MTM analogues with bulky hydrophobic amino acid substitutions on the 3-side chain showed the greatest improvement in PK properties and efficacy. Presumably PK are affected due to increased protein binding and an increase in the pKa, which leads to a lower ionized fraction and decreased uptake in the liver by organic anion transporters. This increase in plasma exposure may lead to increased drug partition in the tumor, as compared to MTM, and, therefore, improved efficacy. Transcriptomic data suggest that the analogs have unique transcriptional effects and therefore unlikely to manifest the same toxicity profile as MTM. Citation Format: Markos Leggas, Ambika Dudhate, Jurgen Rohr. Selective targeting of aberrant ETS transcription factors using uniquely modified mithramycin analogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1851.

Optimizing CSP1 analogs for modulating quorum sensing in Streptococcus pneumoniae with bulky, hydrophobic nonproteogenic amino acid substitutions.

The prompt appearance of multiantibiotic-resistant bacteria necessitates finding alternative treatments that can attenuate bacterial infections while minimizing the rate of antibiotic resistance development. Streptococcus pneumoniae, a notorious human pathogen, is responsible for severe antibiotic-resistant infections. Its pathogenicity is influenced by a cell-density communication system, termed quorum sensing (QS). As a result, controlling QS through the development of peptide-based QS modulators may serve to attenuate pneumococcal infections. Herein, we set out to evaluate the impact of the introduction of bulkier, nonproteogenic side-chain residues on the hydrophobic binding face of CSP1 to optimize receptor-binding interactions in both of the S. pneumoniae specificity groups. Our results indicate that these substitutions optimize the peptide–protein binding interactions, yielding several pneumococcal QS modulators with high potency. Moreover, pharmacological evaluation of lead analogs revealed that the incorporation of nonproteogenic amino acids increased the peptides’ half-life towards enzymatic degradation while remaining nontoxic. Overall, our data convey key considerations for SAR using nonproteogenic amino acids, which provide analogs with better pharmacological properties.

Distinct Mechanisms of Receptor Activation Are Associated with CSF3R Gain-of-Function Mutations

Activating mutations in granulocyte colony-stimulating factor receptor (CSF3R), are found in the extracellular, the membrane proximal, the transmembrane and the cytoplasmic domain of the receptor in various leukemia. To expand the spectrum of CSF3R mutations that can arise to transform Ba/F3 cells initially transfected with wild type CSF3R, we performed a spontaneous cellular transformation assay. We identified several new CSF3R activating mutations. These mutations are located in the extracellular, the membrane proximal, and the transmembrane domains of CSF3R. We further characterized that these mutations transform cells via four distinct mechanisms: (1) cysteine and disulfide bond mediated dimerization, leading to increased STAT3 activation (S581C); (2) polar, non-charged amino acid substitution at the transmembrane helix dimer interface T640, inducing increased STAT3 and ERK activation; (3) increased internalization and STAT3 activation by an E524 substitution; and (4) hydrophobic amino acid substitutions in the membrane proximal O glycosylation sites (T612, T615 and T618), leading to enhanced activation of STAT3 and ERK. Furthermore, we showed that the distinct signaling activation is related to different localization of CSF3R mutants. To this end, we have expanded the region of the CSF3R extracellular and transmembrane domain in which missense mutations exhibit leukemogenic capacity and further elucidated the mechanistic underpinnings that underlie distinct signaling activation and leukemogenic mechanisms. Cumulatively, these findings will facilitate improved evaluation of the biological and clinical relevance of CSF3R mutations identified in patients. [Display omitted] DisclosuresTyner:Agios Pharmaceuticals: Research Funding; Gilead: Research Funding; Syros: Research Funding; Genentech: Research Funding; Constellation Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Janssen Pharmaceutica: Research Funding; Incyte Corporation: Research Funding; Array Biopharma: Research Funding; Takeda Pharmaceutical Company: Research Funding; Aptose Biosciences: Research Funding; Leap Oncology: Consultancy.

The Iodide Transport Defect-Causing Y348D Mutation in the Na+/I- Symporter Renders the Protein Intrinsically Inactive and Impairs Its Targeting to the Plasma Membrane.

Background: The sodium/iodide (Na+/I-) symporter (NIS) mediates active transport of I- into the thyroid gland. Mutations in the SLC5A5 gene, which encodes NIS, cause I- transport defects (ITDs)-which, if left untreated, lead to congenital hypothyroidism and consequent cognitive and developmental deficiencies. The ITD-causing NIS mutation Y348D, located in transmembrane segment (TMS) 9, was reported in three Sudanese patients. Methods: We generated cDNAs coding for Y348D NIS and mutants with other hydrophilic and hydrophobic amino acid substitutions at position 348 and transfected them into cells. The activity of the resulting mutants was quantitated by radioiodide transport assays. NIS glycosylation was investigated by Western blotting after endoglycosidase H (Endo H) and PNGase-F glycosidase treatment. Subcellular localization of the mutant proteins was ascertained by flow cytometry analysis, cell surface biotinylation, and immunofluorescence. The intrinsic activity of Y348D was studied by measuring radioiodide transport in membrane vesicles prepared from Y348D-NIS-expressing cells. Our NIS homology models and molecular dynamics simulations were used to identify residues that interact with Y348 and investigate possible interactions between Y348 and the membrane. The sequences of several Slc5 family transporters were aligned, and a phylogenetic tree was generated in ClustalX. Results: Cells expressing Y348D NIS transport no I-. Furthermore, Y348D NIS is only partially glycosylated, is retained intracellularly, and is intrinsically inactive. Hydrophilic residues other than Asp at position 348 also yield NIS proteins that fail to be targeted to the plasma membrane (PM), whereas hydrophobic residues at this position, which we show do not interact with the membrane, rescue PM targeting and function. Conclusions: Y348D NIS does not reach the PM and is intrinsically inactive. Hydrophobic amino acid substitutions at position 348, however, preserve NIS activity. Our findings are consistent with our homology model's prediction that Y348 should face the side opposite the TMS9 residues that coordinate Na+ and participate in Na+ transport, and with the notion that Y348 interacts only with hydrophobic residues. Hydrophilic or charged residues at position 348 have deleterious effects on NIS PM targeting and activity, whereas a hydrophobic residue at this position rescues NIS activity.

Enhancing the activity and thermal stability of a phthalate-degrading hydrolase by random mutagenesis

Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields.

Feleucin-K3 Analogue with an α-(4-Pentenyl)-Ala Substitution at the Key Site Has More Potent Antimicrobial and Antibiofilm Activities in Vitro and in Vivo.

The development of antimicrobial compounds is now regarded as an urgent problem. Antimicrobial peptides (AMPs) have great potential to become novel antimicrobial drugs. Feleucin-K3 is an α-helical cationic AMP isolated from the skin secretion of the Asian bombinid toad species Bombina orientalis and has antimicrobial activity. In our previous studies, amino acid scanning of Feleucin-K3 was performed to determine the key site affecting its activity. In this study, we investigated and synthesized a series of analogues that have either a natural or an unnatural hydrophobic amino acid substitution at the fourth amino acid residue of Feleucin-K3. Among these analogues, Feleucin-K59 (K59), which has an α-(4-pentenyl)-Ala substitution, was shown to have increased antimicrobial activity against both standard and drug-resistant strains of clinical common bacteria, improved stability, no hemolytic activity at antimicrobial concentrations, and no resistance. In addition, K59 has potent antibiofilm activity in vitro. More importantly, K59 showed better antimicrobial and antibiofilm activities against drug-resistant bacteria in in vivo experiments in mice than traditional antibiotics. In this preliminary study of the mechanism of action, we found that K59 could rapidly kill bacteria by a dual-action mechanism of disrupting the cell membrane and binding to intracellular DNA, thus making it difficult for bacteria to develop resistance.

Flexible RSV Prefusogenic Fusion Glycoprotein Exposes Multiple Neutralizing Epitopes that May Collectively Contribute to Protective Immunity.

Human respiratory syncytial virus (RSV) is a cause of lower respiratory tract infection in infants, young children, and older adults. There is no licensed vaccine and prophylactic treatment options are limited. The RSV fusion (F) glycoprotein is a target of host immunity and thus a focus for vaccine development. F-trimers are metastable and undergo significant rearrangements from the prefusion to a stable postfusion structure with neutralizing epitopes on intermediate structures. We hypothesize that vaccine strategies that recapitulate the breathable F quaternary structure, and provide accessibility of B-cells to epitopes on intermediate conformations, may collectively contribute to protective immunity, while rigid prefusion F structures restrict access to key protective epitopes. To test this hypothesis, we used the near full-length prefusogenic F as a backbone to construct three prefusion F variants with substitutions in the hydrophobic head cavity: (1) disulfide bond mutant (DS), (2) space filling hydrophobic amino acid substitutions (Cav1), and (3) DS, Cav1 double mutant (DS-Cav1). In this study, we compared the immunogenicity of prefusogenic F to prefusion F variants in two animal models. Native prefusogenic F was significantly more immunogenic, producing high titer antibodies to prefusogenic, prefusion, and postfusion F structures, while animals immunized with DS or DS-Cav1 produced antibodies to prefusion F. Importantly, prefusogenic F elicited antibodies that target neutralizing epitopes including prefusion-specific site zero (Ø) and V and conformation-independent neutralizing sites II and IV. Immunization with DS or DS-Cav1 elicited antibodies primarily to prefusion-specific sites Ø and V with little or no antibodies to other key neutralizing sites. Animals immunized with prefusogenic F also had significantly higher levels of antibodies that cross-neutralized RSV A and B subtypes, while immunization with DS or DS-Cav1 produced antibodies primarily to the A subtype. We conclude that breathable trimeric vaccines that closely mimic the native F-structure, and incorporate strategies for B-cell accessibility to protective epitopes, are important considerations for vaccine design. F structures locked in a single conformation restrict access to neutralizing epitopes that may collectively contribute to destabilizing F-trimers important for broad protection. These results also have implications for vaccine strategies targeting other type 1 integral membrane proteins.

Effect of Non-natural Hydrophobic Amino Acids on the Efficacy and Properties of the Antimicrobial Peptide C18G.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules through evolution. The peptides are known to selectively bind to bacterial cell surfaces through electrostatic interactions, and subsequently, the peptides insert into the cell membrane and cause local disruptions of membrane integrity leading to cell death. Previous experiments showed that replacing the Leu residues in the AMP C18G with other naturally occurring hydrophobic residues resulted in side-chain-dependent activities. This work extends the investigation to non-natural hydrophobic amino acids and the effect on peptide activity. Minimal inhibitory concentration (MIC) results demonstrated that amino acid substitutions containing long flexible carbon chains maintained or increased antimicrobial activity compared to natural analogues. In solution, the peptide showed aggregation only with the most hydrophobic non-natural amino acid substitutions. Binding assays using Trp fluorescence confirm a binding preference for anionic lipids while quenching experiments demonstrated that the more hydrophobic peptides are more deeply buried in the anionic lipid bilayers compared to the zwitterionic bilayers. The most effective peptides at killing bacteria were also those which showed some level of disruption of bacterial membranes; however, one peptide sequence exhibited very strong activity and very low levels of red blood cell hemolysis, yielding a promising target for future development.

Protein Translocation Inhibitors Overcome Cytokine-Induced Glucocorticoid Resistance in T-Cell Acute Lymphoblastic Leukemia

Glucocorticoids (GCs) are central to the treatment of T-cell acute lymphoblastic leukemia (T-ALL), and upfront resistance to GCs is a poor prognostic factor. We previously demonstrated that over one-third of primary patient T-ALLs are resistant to the GC dexamethasone (DEX) when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the microenvironment of leukemic blasts and that plays a well-established role in leukemogenesis. Mechanistically, we demonstrated that GCs paradoxically induce their own resistance by promoting the upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augments signal transduction through the JAK/STAT5 axis, ultimately leading to increased STAT5 transcriptional output. This promotes the upregulation of the pro-survival protein BCL-2, which opposes DEX-induced apoptosis. Given that IL7-induced GC resistance depends on de novo synthesis of IL7R in response to DEX, and that newly synthesized IL7R reaches the cell surface via trafficking through the secretory pathway, we hypothesized that inhibiting the translocation of nascent IL7R peptide into the secretory pathway would effectively overcome IL7-induced DEX resistance. Sec61 is a protein-conducting channel in the membrane of the endoplasmic reticulum (ER) that is required for the cotranslational insertion of nascent polypeptides into the ER upon recognition of the signal sequence on secreted and cell surface proteins. To test the hypothesis that Sec61 inhibition could overcome IL7-induced DEX resistance, we utilized the human T-ALL cell line CCRF-CEM, which recapitulates the resistance phenotype observed in primary patient samples. Using a series of structurally distinct small molecule inhibitors of the Sec61 translocon, we demonstrated that Sec61 inhibition effectively overcomes the increase in cell surface IL7R expression in response to DEX. This occurs despite a persistent elevation in IL7R transcript expression following DEX exposure, confirming that Sec61 inhibitors act post-transcriptionally to attenuate cell surface IL7R expression. To determine whether the sensitivity of IL7R to Sec61 inhibitors is due specifically to the interaction between the IL7R signal sequence and Sec61 inhibitors, we generated IL7R constructs containing hydrophobic amino acid substitutions in the signal sequence, which are predicted to confer resistance to Sec61 inhibitors. Upon transient transfection of these constructs into HEK293T cells, we found that these mutations rendered IL7R resistant to the effects of Sec61 inhibition, confirming that the IL7R signal sequence confers sensitivity to these inhibitors. Using the Bliss independence model of synergy in CCRF-CEM cells, we demonstrated that Sec61 inhibitors potently synergize with DEX to overcome IL7-induced DEX resistance. Importantly, at concentrations at which synergy occurs, Sec61 inhibitors demonstrate no single-agent effect on cell survival, suggesting that these effects are not due to an overall reduction in secretory and membrane protein biogenesis. Furthermore, Sec61 inhibitors failed to sensitize CCRF-CEM cells to other chemotherapies used in T-ALL, none of which demonstrate IL7-induced resistance, thereby suggesting that these effects on DEX sensitivity are due specifically to the reduction in cell surface IL7R. To determine if Sec61 inhibitors prevent the DEX-induced increase in STAT5 transcription, we analyzed BCL-2 expression in cells exposed to DEX and IL7, and found that Sec61 inhibitors attenuate the increase in BCL-2 expression in a dose-dependent manner. We next analyzed a cohort of 34 primary patient T-ALL samples. As in CCRF-CEM cells, we found that specifically in those samples with IL7-induced DEX resistance, Sec61 inhibitors synergized with DEX to induce cell death in the presence of IL7. This effect occurred concomitantly with a reduction in cell surface IL7R expression and BCL-2 expression. Taken together, these data demonstrate the efficacy and feasibility of Sec61 inhibition as a novel and rational therapeutic strategy to overcome the IL7-induced DEX resistance phenotype that affects over one-third of newly diagnosed T-ALL patients. Disclosures Sharp: Kezar Life Sciences: Patents & Royalties. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Taunton:Global Blood Therapeutics: Equity Ownership; Principia Biopharma: Equity Ownership, Patents & Royalties; Cedilla Therapeutics: Consultancy, Equity Ownership; Pfizer: Research Funding; Kezar Life Sciences: Equity Ownership, Patents & Royalties, Research Funding.

Hydrophobic Mutations Promote UBQLN2 Oligomerization And Phase Separation

Liquid‐liquid phase separation (LLPS) is hypothesized to be the dominant mechanism that underlies the formation of membraneless organelles, including stress granules formed under cellular stress containing sequestered RNA and proteins. Our lab has recently shown that Ubiquilin‐2 (UBQLN2), a protein involved in protein quality control through proteasomal degradation and autophagy pathways, is recruited to stress granules in vivo and undergoes LLPS in vitro under physiological conditions. Using NMR spectroscopy, we identified both intrinsically‐disordered and folded regions that are involved in UBQLN2 self‐association that also mediate UBQLN2 LLPS. One of these regions includes the proline‐rich (Pxx) segment, where known disease‐linked mutations have been shown to cause 1–2% of familial ALS or ALS/dementia cases. Using size‐exclusion chromatography, NMR spectroscopy, and microscopy, we demonstrated that Pxx mutations modulate UBQLN2 self‐association and phase separation in vitro. Pxx mutations at P497, P506 and P525 affected droplet morphology and dynamics to significantly different extents. Our data suggested that increased hydrophobicity of the amino acid promoted UBQLN2 LLPS and lowered the phase transition temperature at which UBQLN2 LLPS is first observed. To systematically investigate the molecular basis for how amino acid composition affects UBQLN2 phase separation, we generated all 19 possible amino acid replacements at residues P497, P506, P525, and V538. Our data indicate that the phase behavior of UBQLN2 is significantly modulated by mutations at positions 497 and 506, but weakly modulated by mutations at positions 525 and 538. These results suggest that positions 497 and 506 are ‘stickers’ whereas positions 525 and 538 are ‘spacers’ using the language of associative polymers. Consistent with our data above, hydrophobic amino acid substitutions promoted UBQLN2 LLPS. Our experiments suggest that UBQLN2 may be used as a model system to understand the sequence determinants of phase separation. Hydrophobic mutations modulate UBQLN2 oligomerization and LLPS, and potentially alter material properties of UBQLN2‐containing biomolecular condensates in the cell, promoting disease states.Support or Funding InformationC.A.C. graciously acknowledges funding from the ALS Association via grants 17‐IIP‐369 and 18‐IIP‐400, as well as from the National Science Foundation (CAREER award 1750462).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immunoglobulin-like domains of the cargo proteins are essential for protein stability during secretion by the type IX secretion system.

The periodontal pathogen Porphyromonas gingivalis secretes many potent virulence factors using the type IX secretion system (T9SS). T9SS cargo proteins that have been structurally determined by X-ray crystallography are composed of a signal peptide, functional domain(s), an immunoglobulin (Ig)-like domain and a C-terminal domain. Role of the Ig-like domains of cargo proteins in the T9SS has not been elucidated. Gingipain proteases, which are cargo proteins of the T9SS, were degraded when their Ig-like domains were lacking or truncated. The degradation was dependent on the activity of a quality controlfactor, HtrA protease. Another T9SS cargo protein, HBP35, which has a thioredoxin domain as a functional domain, was analyzed by X-ray crystallography, revealing that HBP35 has an Ig-like domain after the thioredoxin domain and that the hydrophobic regions of the thioredoxin domain and the Ig-like domain face each other. HBP35 with substitution of hydrophobic amino acids in the Ig-like domain was degraded depending on HtrA. These results suggest that the Ig-like domain mediates stability of the cargo proteins in the T9SS.

Maturation of the Mfa1 Fimbriae in the Oral Pathogen Porphyromonas gingivalis.

The Mfa1 fimbriae of the periodontal pathogen Porphyromonas gingivalis are involved in adhesion, including binding to synergistic species in oral biofilms. Mfa1 fimbriae are comprised of 5 proteins: the structural component Mfa1, the anchor Mfa2, and Mfa3-5 which constitute the fimbrial tip complex. Interactions among the Mfa proteins and the polymerization mechanism for Mfa1 are poorly understood. Here we show that Mfa3 can bind to Mfa1, 2, 4, and 5 in vitro, and may function as an adaptor protein interlinking other fimbrial subunits. Polymerization of Mfa1 is independent of Mfa3-5 and requires proteolytic processing mediated by the RgpA/B arginine gingipains of P. gingivalis. Both the N- and C- terminal regions of Mfa1 are necessary for polymerization; however, potential β-strand disrupting amino acid substitutions in these regions do not impair Mfa1 polymerization. In contrast, substitution of hydrophobic amino acids with charged residues in either the N- or C- terminal domains yielded Mfa1 proteins that failed to polymerize. Collectively, these results indicate that Mfa3 serves as an adaptor protein between Mfa1 and other accessory fimbrial proteins. Mfa1 fimbrial polymerization is dependent on hydrophobicity in both the N- and C-terminal regions, indicative of an assembly mechanism involving the terminal regions forming a hydrophobic binding interface between Mfa1 subunits.

Gain-of-function mutations in granulocyte colony–stimulating factor receptor (CSF3R) reveal distinct mechanisms of CSF3R activation

Granulocyte colony-stimulating factor (G-CSF or CSF3) and its receptor CSF3R regulate granulopoiesis, neutrophil function, and hematopoietic stem cell mobilization. Recent studies have uncovered an oncogenic role of mutations in the CSF3R gene in many hematologic malignancies. To find additional CSF3R mutations that give rise to cell transformation, we performed a cellular transformation assay in which murine interleukin 3 (IL-3)-dependent Ba/F3 cells were transduced with WT CSF3R plasmid and screened for spontaneous growth in the absence of IL-3. Any outgrowth clones were sequenced to identify CSF3R mutations with transformation capacity. We identified several novel mutations and determined that they transform cells via four distinct mechanisms: 1) cysteine- and disulfide bond-mediated dimerization (S581C); 2) polar, noncharged amino acid substitution at the transmembrane helix dimer interface at residue Thr-640; 3) increased internalization by a Glu-524 substitution that mimics a low G-CSF dose; and 4) hydrophobic amino acid substitutions in the membrane-proximal residues Thr-612, Thr-615, and Thr-618. Furthermore, the change in signaling activation was related to an altered CSF3R localization. We also found that CSF3R-induced STAT3 and ERK activations require CSF3R internalization, whereas STAT5 activation occurred at the cell surface. Cumulatively, we have expanded the regions of the CSF3R extracellular and transmembrane domains in which missense mutations exhibit leukemogenic capacity and have further elucidated the mechanistic underpinnings that underlie altered CSF3R expression, dimerization, and signaling activation.

Effect of Substitutions in Surface Amino Acid on Energy Profile of Apomyoglobin

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0-6 М at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.

Rational design of a low-affinity peptide for the detection of cystatin C in a fast homogeneous immunoassay.

Immunoassays play an essential role in current research and diagnostics resulting in a variety of detection principles. Thereby, homogeneous assays are often used for a fast signal response as demanded for example in point-of-care diagnostics. These systems often rely on a competitive assay design where the sample analyte and the corresponding dye-labeled substance are competing for binding sites on an antibody present in limited amounts. Due to the similar affinities of the antibody towards the sample analyte and the competitor, both sensitivity and assay time are limited. As a consequence, a competitor with a slightly reduced affinity towards the antibody can potentially overcome these drawbacks. Here, we present the rational design of a low-affinity peptide (donor peptide) as a specific analyte competitor for a FRET-based homogeneous immunoassay for the analysis of the protein cystatin C. Thereby, the strategy of peptide-induced antibody generation was combined with the selective variation of the immunization sequence in order to achieve a lower affinity towards the antibody. We could show that shortened donor peptides improved the resulting quenching efficiency in the immunoassay. In addition, the substitution of small hydrophobic amino acids by those with a higher steric demand appeared to be the most promising strategy providing a fast assay response for cystatin C of only 90s.

Tuning gelation kinetics and mechanical rigidity of β-hairpin peptide hydrogels via hydrophobic amino acid substitutions.

Self-assembling peptide hydrogels with faster gelation kinetics and higher mechanical rigidity are favorable for their practical applications. A design strategy to control the folding, self-assembly, and hydrogelation of β-hairpin peptides via hydrophobic amino acid substitutions has been explored in this study. Isoleucine has higher hydrophobicity and stronger propensity for β-sheet hydrogen bonding than valine. After the valine residues of MAX1 (VKVKVKVKV(D)PPTKVKVKVKV-NH2) were replaced with isoleucines, oscillatory rheometry and circular dichroism (CD) spectroscopy characterizations indicated that the variants had clearly faster self-assembly and hydrogelation rates and that the resulting gels displayed higher mechanical stiffness. Transmission electron microscopy (TEM) indicated the parent MAX1 and its variants all formed networks of long and entangled fibrils with the similar diameters of ∼3 nm, suggesting little effect of hydrophobic substitutions on the self-assembled morphology. The MAX1I8 (IKIKIKIKV(D)PPTKIKIKIKI-NH2) hydrogel showed the fastest gelation rate (within 5 min) and the highest gel rigidity with the series, supporting the homogeneous cell distribution within its 3D scaffold. In addition, the MAX1I8 hydrogel showed quick shear-thinning and rapid recovery upon cessation of shear strain, and the MTT and immunological assays indicated its low cytotoxicity and good biocompatibility. These features are highly attractive for its widespread use in 3D cell culturing and regenerative medical treatments.

Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

Structural and Functional Studies on Three Alginate Lyases from Vibrio alginolyticus

To date, several microorganisms that can metabolize alginate have been characterized. In these microorganisms, a class of enzymes called alginate lyases (Alys) catalyze the depolymerization of alginate into oligomers via an endolytic β-elimination reaction. But it is not clear how these lyases differ on their enzymological functions or their divisions of labor. We focused on three extracellular secretory alginate lyases in the Gram-negative marine bacterium V.alginolyticus (ATCC 17749). We found differences in lyase function and catalytic specificity, depending on substrates. These properties apparently derived from demonstrable changes to the protein sequences. We found shared structural domains in alginate lyases specific to the pM and pG substrates through the measurements on enzyme activities and kinetic parameters. Substitution of hydrophobic amino acids in the isoleucine site of domain QIH could have an enormous influence on the high-affinity to pM or pG. This isoleucine was reconfirmed to be indispensable for recognition of the pG or G-G bond. We identified key amino acids located in the catalytic center by means of protein site-directed mutagenesis, and found specific amino acid fragments with exclusive recognitional and catalytic activity effects for different substrates. We reconstructed a series of proteins through splicing, with the goal of producing an engineered protein with the ability to degrade alginate on a variety of substrates and with high enzyme activity as well as temperature stability. Our study provides the theoretical basis for future work on alginate oligosaccharide, transport mechanisms, an area of research with potentially large ramifications for the chemical, medical, textile, printing and agricultural industries.

The Influence of Isoleucine and Arginine on Biological Activity and Peptide- Membrane Interactions of Antimicrobial Peptides from the Bactericidal Domain of AvBD4

In this study, the influence of isoleucine and arginine on the biological activity and peptide-membrane interactions of linear avian β-defensin-4 (RL38) analogs was investigated. Results of biological activities showed that the antimicrobial activities of AvBD-4 analogs were closely related to hydrophobicity and amphipathicity. The peptide GLI19 with high hydrophobicity value and amphipathicity displayed broad spectrum antimicrobial activity against both gram-negative and gram-positive, whereas GLR19 with increasing multiple charges only exhibited activity against gram-negative. The interaction between peptides and the liposome membrane demonstrated that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which supported the antimicrobial activity data. The outer membranes assay further demonstrated that GLI19 had a greater capacity than the other tested peptides to penetrate the cell membrane at a low concentration. Collectively, the peptides derived from the bactericidal domain of linear β- defensins by truncation and hydrophobic amino acid substitution may be effective high-potential antibacterial agents.