Single Amino Acid Substitution Mutants Research Articles

Abstract 377: Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function

Abstract Introduction: KEAP1 and NFE2L2 (NFR2) mutations occur in roughly 20% of non-small cell lung cancer (NSCLC) patients and are associated with resistance to a broad range of therapies including radiotherapy, chemotherapy, targeted therapies, and immunotherapy. In prior work, we demonstrated that not all KEAP1 mutations observed in NSCLC patients are pathogenic. These benign mutations have a wild-type phenotype that does not impart therapy resistance. However, only roughly 100 of ~13,000 (<1%) possible KEAP1 single amino acid substitution mutations have been characterized. Here, we performed saturation mutagenesis of KEAP1 to comprehensively evaluate all possible point mutation phenotypes in vitro. Methods: Saturation mutagenesis was accomplished using the High-throughput Mutagenesis by Integrated TilEs (MITE) method. In brief, the KEAP1 sequence was divided into 21 tiles each containing a 90 bp variable region flanked by a 30 bp constant region. At each codon position, mutations representing the 20 possible natural amino acids and a stop codon were included for a total of 13,076 KEAP1 variants. The tiles were assembled such that each full length KEAP1 construct contained a single desired mutation. The KEAP1 MITE lentivirus library was transduced into H1299-KEAP1NULL cells that were then treated with vehicle or H2O2 for 24 days. The KEAP1 ORF was amplified from gDNA and mutations were quantified by next generation sequencing. Mutation enrichment analysis was used to derive functional annotations. Results: Oxidative stress resulted in a bimodal distribution of KEAP1 mutations. Cells with KEAP1 nonsense mutations were enriched while cells with silent mutations were depleted. Using a threshold of 95% specificity for silent mutations to distinguish benign from pathogenic mutations, 94% of nonsense mutations and 88% of previously characterized pathogenic mutations were identified as pathogenic. While the majority of KEAP1 mutations were benign (64%), analysis of mutations found in TCGA NSCLC patients showed an enrichment of pathogenic mutations (72%). Overlaying mutation calls onto the KEAP1 structure showed enrichment of pathogenic mutations in the BTB homodimerization interface, hydrophobic core of the oxidative stress sensing IVR domain, and NRF2 binding pocket of the Kelch domain. Conclusions: Saturation mutagenesis of KEAP1 generated a comprehensive list of pathogenic KEAP1 single amino acid substitutions. Assignments strongly agreed with biologic function and prior literature. The assessment of KEAP1 mutation phenotypes is important as benign mutations occur in a significant subset of NSCLC patients and are not expected to impart therapy resistance. Our results will allow identification of benign and pathogenic KEAP1 mutations in clinical studies, which will enable testing of personalized therapeutic strategies for patients with KEAP1 mutations. Citation Format: Noah Kastelowitz, Shashank Shrishrimal, Soyeong Jun, Anni M. Zhang, Rui Wang, Ilayda Ilerten, Maximilian Diehn. Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 377.

Evolutionary Conservation of PP2A Antagonism and G2/M Cell Cycle Arrest in Maedi-Visna Virus Vif.

The canonical function of lentiviral Vif proteins is to counteract the mutagenic potential of APOBEC3 antiviral restriction factors. However, recent studies have discovered that Vif proteins from diverse HIV-1 and simian immunodeficiency virus (SIV) isolates degrade cellular B56 phosphoregulators to remodel the host phosphoproteome and induce G2/M cell cycle arrest. Here, we evaluate the conservation of this activity among non-primate lentiviral Vif proteins using fluorescence-based degradation assays and demonstrate that maedi-visna virus (MVV) Vif efficiently degrades all five B56 family members. Testing an extensive panel of single amino acid substitution mutants revealed that MVV Vif recognizes B56 proteins through a conserved network of electrostatic interactions. Furthermore, experiments using genetic and pharmacologic approaches demonstrate that degradation of B56 proteins requires the cellular cofactor cyclophilin A. Lastly, MVV Vif-mediated depletion of B56 proteins induces a potent G2/M cell cycle arrest phenotype. Therefore, remodeling of the cellular phosphoproteome and induction of G2/M cell cycle arrest are ancient and conserved functions of lentiviral Vif proteins, which suggests that they are advantageous for lentiviral pathogenesis.

A unique self-truncation of bacterial GH5 endoglucanases leads to enhanced activity and thermostability

Backgroundβ-1,4-endoglucanase (EG) is one of the three types of cellulases used in cellulose saccharification during lignocellulosic biofuel/biomaterial production. GsCelA is an EG secreted by the thermophilic bacterium Geobacillus sp. 70PC53 isolated from rice straw compost in southern Taiwan. This enzyme belongs to glycoside hydrolase family 5 (GH5) with a TIM-barrel structure common among all members of this family. GsCelA exhibits excellent lignocellulolytic activity and thermostability. In the course of investigating the regulation of this enzyme, it was fortuitously discovered that GsCelA undergoes a novel self-truncation/activation process that appears to be common among GH5 enzymes.ResultsThree diverse Gram-positive bacterial GH5 EGs, but not a GH12 EG, undergo an unexpected self-truncation process by removing a part of their C-terminal region. This unique process has been studied in detail with GsCelA. The purified recombinant GsCelA was capable of removing a 53-amino-acid peptide from the C-terminus. Natural or engineered GsCelA truncated variants, with up to 60-amino-acid deletion from the C-terminus, exhibited higher specific activity and thermostability than the full-length enzyme. Interestingly, the C-terminal part that is removed in this self-truncation process is capable of binding to cellulosic substrates of EGs. The protein truncation, which is pH and temperature dependent, occurred between amino acids 315 and 316, but removal of these two amino acids did not stop the process. Furthermore, mutations of E142A and E231A, which are essential for EG activity, did not affect the protein self-truncation process. Conversely, two single amino acid substitution mutations affected the self-truncation activity without much impact on EG activities. In Geobacillus sp. 70PC53, the full-length GsCelA was first synthesized in the cell but progressively transformed into the truncated form and eventually secreted. The GsCelA self-truncation was not affected by standard protease inhibitors, but could be suppressed by EDTA and EGTA and enhanced by certain divalent ions, such as Ca2+, Mg2+, and Cu2+.ConclusionsThis study reveals novel insights into the strategy of Gram-positive bacteria for directing their GH5 EGs to the substrate, and then releasing the catalytic part for enhanced activity via a spontaneous self-truncation process.

The base and root of domain II loops of Cry toxins contribute to binding to Bombyx mori ABC transporter C2.

Little information is available regarding the region of Cry toxins involved in binding to their major receptors, the ATP-binding cassette (ABC) transporters. We analyzed which Cry1Aa amino acid residues contribute to binding to Bombyx mori ABC transporter C2 (BmABCC2). Several two oxidized double-cysteine substitution mutant toxins were made. In these, two amino acids at distant positions on toxin loop α8 and loop 2 or loop 2 and loop 3 were substituted with cysteine residues and crosslinked. These mutants exhibited a marked reduction in binding affinity to BmABCC2, suggesting that the binding site comprises complex cavities formed by loops α8, 2, and 3. Loop swapping between Cry1Aa and other BmABCC2-incompatible toxins indicated that loop 2 acts as a binding affinity-generating part of Cry1Aa toxin. Using single amino acid substitution mutants, the results of surface plasmon resonance (SPR) analysis and response assays with BmABCC2-expressing Sf9 cells indicated that Y366, R367, R368, and L447 in the Cry1Aa root and base region of loops 2 and 3 play important roles in binding. Furthermore, SPR analyses of these mutants suggested that a two-state binding model fits best the data obtained. Moreover, complex cavities and the above-mentioned amino acid residues contribute to the generation of multiple binding points and high-affinity binding. Finally, we found that the binding site of B. mori cadherin-like protein consists of complex cavities comprising loops 1, 2, and 3, partially overlapping that of BmABCC2, suggesting that the loop region of Cry1Aa toxin acts as a promiscuous binding site.

Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine

Aegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The most studied fungal aegerolysins share a common property of interacting with membranes enriched with cholesterol in combination with either sphingomyelin or ceramide phosphorylethanolamine (CPE), major sphingolipids in the cell membranes of vertebrates and invertebrates, respectively. However, genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibrio; these are human pathogens of high clinical relevance and can thrive in a variety of other species. The knowledge on bacterial aegerolysin-lipid interactions is scarce. We show that Pseudomonas aeruginosa aegerolysin RahU interacts with CPE, but not with sphingomyelin-enriched artificial membranes, and that RahU interacts with the insect cell line producing CPE. We report crystal structures of RahU alone and in complex with tris(hydroxymethyl)aminomethane (Tris), which, like the phosphorylethanolamine head group of CPE, contains a primary amine. The RahU structures reveal that the two loops proximal to the amino terminus form a cavity that accommodates Tris, and that the flexibility of these two loops is important for this interaction. We show that Tris interferes with CPE-enriched membranes for binding to RahU, implying on the importance of the ligand cavity between the loops and its proximity in RahU membrane interaction. We further support this by studying the interaction of single amino acid substitution mutants of RahU with the CPE-enriched membranes. Our results thus represent a starting point for a better understanding of the role of P. aeruginosa RahU, and possibly other bacterial aegerolysins, in bacterial interactions with other organisms.

Functional information from clinically-derived drug resistant forms of the Candida glabrata Pdr1 transcription factor.

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.

Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization.

The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.

Recent developments in isotope-aided NMR methods for supramolecular protein complexes –SAIL aromatic TROSY

BackgroundThe structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. MethodsThe 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. ResultsThe MSG proteins, selectively labeled with either [δ1,ε1,ε3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. ConclusionsWe demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. General significanceThe method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.

Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2

The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs. A recent study identified neural epidermal growth factor-like (NEL)-like 2 (NELL2) as a novel ligand for Robo3. In this study, we carried out a comprehensive analysis of the interaction between NELL1 and the Robo family of receptors and demonstrated that Robo2 contains a cryptic binding site for both NELL1 and NELL2. NELL1/2 binds to the first fibronectin type III (FNIII) domain of Robo2 but not to intact Robo2. Mutation analysis revealed that several amino acids within the first FNIII domain are critical for NELL1 binding to Robo2 but not to Robo1. The Robo2 deletion mutants without the fourth immunoglobulin domain and single amino acid substitution mutants that can influence the architecture of the ectodomain facilitated binding to NELL1/2. Acidic conditions increased the binding affinity of Robo2 for NELL1. These results suggest that Robo2 functions as a receptor for NELL1/2, particularly under circumstances where Robo2 undergoes proteolytic digestion. If this is not the case, conformational changes of the ectodomain of Robo2 may unmask the binding site for NELL1/2.

F331H mutation and reduced mRNA in type-1 acetylcholinesterase is associated with carbofuran resistance development in the small brown planthopper (Laodelphax striatellus Fallén)

Molecular diagnostic markers are necessary for establishing high-throughput screening systems to support insecticide-resistant population management. Here, we identified single amino acid substitution mutations related to carbamate resistance in Laodelphax striatellus Fallén type-1 acetylcholinesterase (LsAChE1) using carbofuran-selected strains. The phenotypic resistance profiles of the final selection strain (SEL9) compared to the susceptible strain revealed a 14-fold higher resistance ratio based on topical application, 1.2-fold higher general esterase activity, and 4.3-fold higher acetylcholinesterase insensitivity based on the 50% inhibitory concentration (I50), suggesting that insensitivity of the target site could occur as a resistance factor. Comparison of the nucleotide sequences of Lsace1 of five strains (SUS, SEL0, SEL3, SEL6, and SEL9) revealed two amino acid substitutions (F330Y and F331H). To understand the roles of these mutations, we determined the allele frequency of both point mutations in the selected strains using quantitative sequencing methods. In addition, several quantitative genotypic traits (e.g., gene copy numbers and transcript levels of Lsace1, Lsace2, and LS.CarE1) were assessed. A correlation analysis of genotypic and phenotypic traits revealed strong correlations between resistance level and I50 with F331H allele frequency. Interestingly, the F331H mutation was negatively correlated with transcript levels of Lsace1, suggesting that selection pressure might result in a reduction of the target gene. Overall, the F331H mutation and reduced mRNA are important factors in the development of carbamate resistance. Furthermore, the point mutation can be used to monitor rapid carbofuran resistance in conjunction with molecular diagnostic methods such as quantitative sequencing.

Abstract 394: Saturation mutagenesis of KRAS reveals the functional landscape of missense variants

Abstract KRAS is the most commonly mutated oncogenes and is a major driver of tumor initiation and progression. Understanding the functional consequences of cancer-associated KRAS variants may have important clinical implications. For example, KRAS mutation status defines those that are likely to respond to EGFR-directed therapy in KRAS-mutant metastatic colorectal cancer. A compendium of all possible oncogenic KRAS alleles would serve as a roadmap for future therapeutic strategies directed at KRAS itself or downstream signaling effectors. Comprehensive mutagenesis of KRAS may also elucidate structure-function relationships that reveal novel biochemical properties that may be exploited for therapeutic gain. We performed saturation mutagenesis of both a wild-type (WT) and a G12D mutant form of KRAS cDNA and generated lentiviral expression libraries of 3,553 and 3,534 single amino acid substitution mutants of each backbone. We utilized these WT and G12D mutagenesis libraries for functional genetic screening to identify gain- and loss-of-function missense variants that alter critical oncogenic properties of KRAS. First, we sought to comprehensively identify all possible oncogenic missense mutations in KRAS that mediate oncogenic transformation. We stably transduced the WT library into immortalized human epithelial cells and evaluated growth in low attachment (GILA), an assay that is highly correlated with in vivo tumor formation. We identified all previously known hotspot oncogenic alleles of KRAS as well as many functionally relevant alleles that are also discovered at lower frequency in human tumors. Moreover, we also discovered a group of transforming KRAS variants that have not been well described in human tumors, thus revealing potentially novel activating mechanisms for oncogenic KRAS. In parallel, we utilized the G12D mutagenesis library to perform second-site suppressor screening to identify loss-of-function single amino acid changes that abrogate the transforming ability of oncogenic KRAS. We performed positive-selection screening in primary cell lines for variants that enable bypass of oncogene-induced senescence. Additionally, we conducted a negative-selection screen with the G12D library in a KRAS-dependent cancer cell line with inducible suppression of endogenous KRAS, thus identifying all possible second-site mutations that abolish KRAS-driven signaling necessary for maintenance of cellular proliferation and viability. Structure-function analysis of these data may reveal novel patterns of amino-acid changes that result in inactivation of oncogenic KRAS. In summary, this comprehensive dictionary of gain- and loss-of-function KRAS mutants will facilitate understanding of clinically important mutations and also yield novel insights into structure-function relationships that may improve our understanding of the KRAS oncogene. Citation Format: Eejung Kim, Seav Huong Ly, Nicole S. Persky, Belinda Wang, Xiaoping Yang, Federica Piccioni, Katherine Labella, Mihir Doshi, Robert E. Lintner, Cong Zhu, Scott Steelman, David E. Root, Cory M. Johannessen, Alex B. Burgin, Laura E. MacConaill, William C. Hahn, Andrew J. Aguirre. Saturation mutagenesis of KRAS reveals the functional landscape of missense variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2017-394

Fine mapping and candidate gene analysis of a novel PANICLE AND SPIKELET DEGENERATION gene in rice

To better understand the molecular mechanisms that regulate inflorescence and flower architecture, we characterized the rice panicle and spikelet degeneration (psd) mutant, which exhibited small panicles with a reduced number of primary and secondary branches. The psd mutant had few spikelets per panicle, and they were deformed and could not form seeds. The psd mutant had a unique panicle organization phenotype that was not present in other aberrant panicle organization mutants. Genetic analysis revealed that the mutation was controlled by a single recessive gene. A map-based cloning strategy was employed to isolate the PSD locus. The PSD locus was initially located between RM13046 and RM5356 on chromosome 2. Recessive individuals (n = 7307) in F2 mapping populations from two crosses between heterozygous Psdpsd plants and two indica rice varieties, IR28 and 93-11, were used to finely map the PSD locus. The PSD locus was mapped to a 14.6 kb region between two newly developed dCAPS markers, dC7 and dC8, within BAC OJ1115_D03. The candidate regions harbored two ORFs, Os02g0284500 and Os02g0284600. Sequence analysis revealed that only one single amino acid substitution mutation in the Os02g0284500 gene in the candidate region, which altered the predicted three-dimensional Os02g0284500 protein structure. A tetra-primer amplification refractory mutation system-PCR marker, TP-psd, was developed from the psd mutant mutation site. All 7307 homozygote recessive plants in the two F2 populations showed the same band pattern as that of psdpsd. This suggested that Os02g0284500 is most likely the PSD gene. Our results provide a basis for future functional PSD studies.

Mutant screening for oncogenes of Ewing's sarcoma using yeast.

Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast. In this screening, we identified 13 single amino acid substitution mutants which could suppress growth inhibition by oncogenes. All of the point mutation positions of the EWS/ETS family proteins were located within the ETS domain, which is responsible for the interaction with a specific DNA motif. Eight-mutated residues within the ETS domain matched to 13 completely conserved amino acid residues in the human ETS domains. Moreover, mutants also showed reduced transcriptional activities on the DKK2 promoter, which is upregulated by the EWS/ETS family, compared to that of the wild type. These results suggest that the ETS domain in the EWS/ETS family proteins may be a primary target for growth inhibition of Ewing's sarcoma and that this yeast screening system can be applied for the functional screening of the oncogenes.

The many faces of prolactin in breast cancer.

Prolactin (PRL) is a neuroendocrine polypeptide hormone primarily produced by the lactotrophs in the anterior pituitary gland of all vertebrates. The physiological role of PRL in mammary glands is relatively certain while its role in breast tumor has been a topic of debate for over 20 years. In this review, the author attempts to briefly summarize the data coming from his laboratory in the past years, focusing on G129R, a PRL receptor (PRLR) antagonist developed by introducing a single amino acid substitution mutation into human PRL (hPRL) at position 129, and a variety of G129R derivatives. The author has proposed two novel ideas for potential use of PRL, not anti-PRL agents, as an adjuvant agent for breast cancer, making it a hormone of many faces.

Biochemical Analysis of DNA Polymerase η Fidelity in the Presence of Replication Protein A

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify in hosts. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To probe the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the efficient and proper assembly of a mature viral capsid. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, some mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies.

Substrate profiling of cyclohexylamine oxidase and its mutants reveals new biocatalytic potential in deracemization of racemic amines

A cyclohexylamine oxidase (CHAO) of bacterial origin was previously shown to be a potentially useful catalyst in the deracemization of racemic primary amines. To further explore the properties and application of this enzyme, five single-amino acid substitution mutants (L199A, M226A, Y321A, Y321F, and L353M) were created based on superimposition of the tertiary structure of CHAO and the monoamine oxidase (MAO) B homolog. The substrate specificity of the purified wild-type and five mutant enzymes were examined towards 38 structurally diverse amines. All the enzymes exhibited better activity for primary amines than secondary and tertiary amines and in general exhibited high stereoselectivity. Among the mutant enzymes, M226A displayed an enhanced activity (5-400%) towards most substrates, and L353M showed 7-445% higher activity towards primary aliphatic amines with cycloalkane or aromatic moieties. Kinetic parameters revealed that both Y321 mutants showed higher catalytic efficiency towards cyclooctanamine, whereas the wild-type CHAO (wt CHAO) was most efficient towards cyclohexylamine. The wt CHAO or variant L353M in combination with a borane-ammonia complex as reducing agent was applied to the deracemization of 1-aminotetraline to give the (R)-enantiomer, a precursor of an antidepressant drug Norsertraline, in good yield (73-76%), demonstrating their application potential in chiral amine synthesis.

Extreme Genetic Fragility of the HIV-1 Capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies.

The Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae Binds to Lipid Raft Microdomains in Human Corneal Epithelial Cells

Streptococcus pneumoniae (pneumococcus) is an opportunistic bacterial pathogen responsible for causing several human diseases including pneumonia, meningitis, and otitis media. Pneumococcus is also a major cause of human ocular infections and is commonly isolated in cases of bacterial keratitis, an infection of the cornea. The ocular pathology that occurs during pneumococcal keratitis is partly due to the actions of pneumolysin (Ply), a cholesterol-dependent cytolysin produced by pneumococcus. The lytic mechanism of Ply is a three step process beginning with surface binding to cholesterol. Multiple Ply monomers then oligomerize to form a prepore. The prepore then undergoes a conformational change that creates a large pore in the host cell membrane, resulting in cell lysis. We engineered a collection of single amino acid substitution mutants at residues (A370, A406, W433, and L460) that are crucial to the progression of the lytic mechanism and determined the effects that these mutations had on lytic function. Both PlyWT and the mutant Ply molecules (PlyA370G, PlyA370E, PlyA406G, PlyA406E, PlyW433G, PlyW433E, PlyW433F, PlyL460G, and PlyL460E) were able to bind to the surface of human corneal epithelial cells (HCECs) with similar efficiency. Additionally, PlyWT localized to cholesterol-rich microdomains on the HCEC surface, however, only one mutant (PlyA370G) was able to duplicate this behavior. Four of the 9 mutant Ply molecules (PlyA370E, PlyW433G, PlyW433E, and PlyL460E) were deficient in oligomer formation. Lastly, all of the mutant Ply molecules, except PlyA370G, exhibited significantly impaired lytic activity on HCECs. The other 8 mutants all experienced a reduction in lytic activity, but 4 of the 8 retained the ability to oligomerize. A thorough understanding of the molecular interactions that occur between Ply and the target cell, could lead to targeted treatments aimed to reduce the pathology observed during pneumococcal keratitis.

Site-Specific Cleavage of the Host Poly(A) Binding Protein by the Encephalomyocarditis Virus 3C Proteinase Stimulates Viral Replication

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.