Introduction Of Point Mutations Research Articles

In vivoandin vitrodisease modeling with CRISPR/Cas9

In the past few years, extensive progress has been made in the development of genome-editing technology. Among several genome-editing tools, the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) system is particularly widely used owing to the ease of sequence-specific nuclease construction and the highly efficient introduction of mutations. The CRISPR/Cas9 system was originally constructed to induce small insertion and deletion mutations, but various methods have been developed to introduce point mutations, deletions, insertions, chromosomal translocations and so on. These methods should be useful for the reconstruction of disease-causing mutations in cultured cell lines and living organisms to elucidate disease pathogenesis and for disease prevention, treatment and drug discovery. This review summarizes the current technical aspects of the CRISPR/Cas9 system for disease modeling in cultured cells and living organisms, mainly mice.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194(ts) for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter PmanP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168. In prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

9. rAAV Designs Harboring DNA Secondary Structures with High Thermal Stabilities Produce Heterogenic Viral Genome Populations

It has been known for more than a decade that AAV genomes in ssAAV particles are heterogeneous, and can contain smaller than unit-length molecules to varying degrees. However, the cause underlying this phenomenon remained unknown. Our earlier work demonstrated that short DNA hairpin (shDNA) sequences designed into rAAV vectors, resulted in truncated genomes through a template-switching mechanism during AAV genome replication. Importantly, the knowledge we have gained about shDNA-mediated genomic truncation has helped to improve shRNA-cassette design. Specifically, we have found a correlation between the thermal stability of shDNA structures with the prevalence of truncation events. By introducing point mutations into the passenger strand of shRNA sequences to create DNA bulges within shDNAs in scAAV vectors, we found that lowering the thermal stability of DNA hairpins lowered the proportion of truncated to complete genomes. In addition, we found that scAAV vectors incorporated with pri-miRNA transgenes, which contain natural bulges in their stem-loop structures, also produced fewer truncated genomes, relative to shDNA-rAAV constructs. By embedding the guide strand of small RNAs into pri-miRNA scaffolds, we have defined critical improvements to the genomic integrity of rAAV vectors expressing small RNAs. Furthermore, we developed a method, named AAV-GPSeq (AAV genome populations sequencing), to directly sequence whole vector genome populations from purified rAAVs using the PacBio platform for high-throughput sequencing. We discovered through this methodology that truncation events can originate from palindromic sequences and inverted repeats that reside in the expression cassette elements of widely used ssAAV and scAAV vector designs (i.e. transcriptional regulatory elements, transgene sequences, and post-transcriptional regulatory regions). The resulting diversity of genomic truncations produces populations of packaged virions with variable transgene efficacies, and gives us the first insights into the phenomenon of rAAV heterogeneity. By examining the genomes from rAAV vectors harboring shDNAs, pri-miRNA fragments, and palindromic/inverted repeat sequences, we now highlight the importance of DNA secondary structure on vector design and genomic heterogeneity in packaged viral vectors. Improvements upon quality control standards are thus necessary and critical to efficacious and safe clinical uses for rAAV as a biomedicine. Further implications for our novel findings towards understanding AAV replication, and new considerations for future therapeutic rAAV vector designs will be discussed. To improve the homogeneity of clinical rAAV vectors, we are optimizing the vector production procedure, testing non-palindrome promoters, changing the codon usage in transgenes to lower the thermostability of rAAV genomes, and modifying rAAV packaging cell lines to minimize template-switching events to produce more intact rAAV genomes.

Seamless site-directed mutagenesis of the Saccharomyces cerevisiae genome using CRISPR-Cas9.

CRISPR assisted homology directed repair enables the introduction of virtually any modification to the Saccharomyces cerevisiae genome. Of obvious interest is the marker-free and seamless introduction of point mutations. To fulfill this promise, a strategy that effects single nucleotide changes while preventing repeated recognition and cutting by the gRNA/Cas9 complex is needed. We demonstrate a two-step method to introduce point mutations at 17 positions in the S. cerevisiae genome. We show the general applicability of the method, enabling the seamless introduction of single nucleotide changes at any location, including essential genes and non-coding regions. We also show a quantifiable phenotype for a point mutation introduced in gene GSH1. The ease and wide applicability of this general method, combined with the demonstration of its feasibility will enable genome editing at an unprecedented level of detail in yeast and other organisms.

Mutant bacterial sodium channels as models for local anesthetic block of eukaryotic proteins

ABSTRACTVoltage gated sodium channels are the target of a range of local anesthetic, anti-epileptic and anti-arrhythmic compounds. But, gaining a molecular level understanding of their mode of action is difficult as we only have atomic resolution structures of bacterial sodium channels not their eukaryotic counterparts. In this study we used molecular dynamics simulations to demonstrate that the binding sites of both the local anesthetic benzocaine and the anti-epileptic phenytoin to the bacterial sodium channel NavAb can be altered significantly by the introduction of point mutations. Free energy techniques were applied to show that increased aromaticity in the pore of the channel, used to emulate the aromatic residues observed in eukaryotic Nav1.2, led to changes in the location of binding and dissociation constants of each drug relative to wild type NavAb. Further, binding locations and dissociation constants obtained for both benzocaine (660 μM) and phenytoin (1 μ M) in the mutant channels were within the range expected from experimental values obtained from drug binding to eukaryotic sodium channels, indicating that these mutant NavAb may be a better model for drug binding to eukaryotic channels than the wild type.

SGK1 regulation by miR-466g in cortical collecting duct cells.

Micro-RNAs (miRNAs) are noncoding RNAs that bind target mRNA transcripts and modulate gene expression. In the cortical collecting duct (CCD), aldosterone stimulates the expression of genes that increase activity of the epithelial sodium channel (ENaC); in the early phase of aldosterone induction, one such gene is serum and glucocorticoid regulated kinase 1 (SGK1). We hypothesized that aldosterone regulates the expression of miRNAs in the early phase of induction to control the expression of target genes that stimulate ENaC activity. We treated mpkCCDc14 cells with aldosterone or vehicle for 1 h and used a miRNA microarray to analyze differential miRNA expression. We identified miR-466g as a miRNA that decreased by 57% after 1 h of aldosterone treatment. Moreover, we identified a putative miR-466g binding site in the 3'-untranslated region of SGK1. We constructed an SGK1 3'-untranslated region luciferase reporter and found that cotransfection of miR-466g suppressed luciferase activity in human embryonic kidney-293 cells in a dose-dependent manner. Deletion or introduction of point mutations that disrupt the miR-466g target site attenuated miR-466g-directed suppression of luciferase activity. Finally, we generated stably transduced mpkCCDc14 cell lines overexpressing miR-466g. Cells overexpressing miR-466g demonstrated 12.9-fold lower level of SGK1 mRNA compared with control cells after 6 h of aldosterone induction; moreover, cells overexpressing miR-466g exhibited 25% decrease in amiloride-sensitive current after 6 h of aldosterone induction and complete loss of amiloride-sensitive current after 24 h of aldosterone induction. Our findings implicate miR-466g as a novel early-phase aldosterone responsive miRNA that regulates SGK1 and ENaC in CCD cells.

A Charged Residue in the HCN Channel C-Linker Stabilizes the Open State

Hyperpolarization- and cyclic nucleotide-activated (HCN) channels are regulated by a voltage sensing domain (VSD) as well as a cyclic nucleotide binding domain (CNBD) coupled to the pore by a C-linker. We previously found a mutation in the VSD that slows deactivation kinetics by creating a highly stable open state (Open State Trapping, OST) that is dependent on cAMP binding; deletion of the CNBD and C-linker (ΔC) completely removed OST. I introduced point mutations in the C-linker in an OST mutant, and tested their effects on the stable open conformation. Deactivation kinetics of the mutants, and the ΔC construct, were studied in Xenopus laevis oocytes using two electrode voltage clamp, and fitted to an exponential decay equation. Arginine substitution of a conserved glutamate significantly decreased the deactivation time constant (τ) at +20 mV by ∼3.5 fold when compared to wild type. The same mutation shifted the isochronal deactivation midpoint voltage (Vdeact) by −41 mV. For ΔC, ΔVdeact was −55 mV relative to wild type and τ was ∼8 fold faster than wild type. Therefore, the charge reversal mutation removed a substantial portion of the OST effect on τ and the majority of the OST effect on Vdeact. In comparison, mutating the same residue to glutamine or cysteine partially removed the OST effects, with τ decreased by ∼1.5 fold and 3 fold respectively, and with ΔVdeact of −9 mV and −19 mV respectively. We conclude the native glutamate stabilizes the open state during OST, possibly due to C-linker interactions with other channel regions. Future research will explore how and when this stabilization occurs, for example if it can still occur in the absence of cAMP binding or of the OST mechanism.

Controlling the transcription levels of argGH redistributed L-arginine metabolic flux in N-acetylglutamate kinase and ArgR-deregulated Corynebacterium crenatum.

Corynebacterium crenatum SYPA5-5, an L-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits L-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by L-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (L-citrulline and L-ornithine) and decreased production of L-arginine. Genetic and enzymatic levels analysis involved in L-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from L-citrulline to L-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased L-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the L-arginine productivity improved about 63.6%. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM L-arginine with the productivity of 5.42 mM h(-1). These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward L-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for L-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct L-arginine high-producing C. crenatum.

Highly efficient and easy protease-mediated protein purification.

As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate.

Mechanism of Action of ME1111, a Novel Antifungal Agent for Topical Treatment of Onychomycosis

Despite the existing treatment options for onychomycosis, there remains a strong demand for potent topical medications. ME1111 is a novel antifungal agent that is active against dermatophytes, has an excellent ability to penetrate human nails, and is being developed as a topical agent for onychomycosis. In the present study, we investigated its mechanism of action. Trichophyton mentagrophytes mutants with reduced susceptibility to ME1111 were selected in our laboratory, and genome sequences were determined for 3 resistant mutants. The inhibitory effect on a candidate target was evaluated by a spectrophotometric enzyme assay using mitochondrial fractions. Point mutations were introduced into candidate genes by a reverse genetics approach. Whole-genome analysis of the 3 selected mutants revealed point mutations in the structural regions of genes encoding subunits of succinate dehydrogenase (complex II). All of the laboratory-generated resistant mutants tested harbored a mutation in one of the subunits of succinate dehydrogenase (SdhB, SdhC, or SdhD). Most of the mutants showed cross-resistance to carboxin and boscalid, which are succinate dehydrogenase inhibitors. ME1111 strongly inhibited the succinate-2,6-dichlorophenolindophenol reductase reaction in Trichophyton rubrum and T. mentagrophytes (50% inhibitory concentrations [IC50s] of 0.029 and 0.025 μg/ml, respectively) but demonstrated only moderate inhibition of the same reaction in human cell lines. Furthermore, the target protein of ME1111 was confirmed by the introduction of point mutations causing the amino acid substitutions in SdhB, SdhC, and SdhD found in the laboratory-generated resistant mutants, which resulted in reduced susceptibility to ME1111. Thus, ME1111 is a novel inhibitor of the succinate dehydrogenase of Trichophyton species, and its mechanism of action indicates its selective profile.

CRISPR/Cas9-Mediated Rapid Generation of Multiple Mouse Lines Identified Ccdc63 as Essential for Spermiogenesis.

Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.

Abstract 2036: Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth

Abstract One of the most fundamental traits of cancer cells is the ability to sustain proliferation by circumventing cell cycle regulatory programs normally controlled by tumor suppressors. Keratin 17 (K17) is not expressed in most somatic tissues but is overexpressed and is a negative prognostic marker in cervical, breast, ovarian, and gastric carcinomas. The mechanisms by which K17 contributes to cancer-related signaling, however, remain unknown. Here, we report that nuclear-localized K17 interacts with tumor suppressor p27KIP1 and mediates its nuclear export, maintaining proliferation. After validating K17 is a prognostic marker in cervical cancer, independent of tumor stage, we performed in vitro and in vivo experiments to investigate the role of K17 in proliferative signaling and tumor growth using loss- and gain- of function approaches in cervical cancer and other cancer-derived cell lines. We found that K17 expression maintains proliferation, while silencing K17 induces G1 arrest by nuclear accumulation and stabilization of tumor suppressors p27KIP1 and pRb. During G1, K17 localizes in the nucleus, mediated by a classical bipartite nuclear localization signal (NLS) (AA385-400) that was identified by in silico analysis. This NLS is specific among keratins and present only in primates but not in other species. To our knowledge, this is the first report that a keratin has a NLS and is capable of undergoing nuclear translocation. During G1, p27KIP1is exported from the nucleus in a CRM1-dependent manner and is subsequently degraded, triggering G1/S transition. p27KIP1 lacks the classical leucine-rich nuclear export signal (NES) and requires an adaptor for CRM1-exportin binding. After confirming the binding of K17 and p27KIP1 within the nucleus, we performed in silico analyses and identified a leucine-rich NES required for CRM1-binding in K17 (AA194-199), which was further validated by a 3-fold nuclear retention of K17 and p27KIP1 after leptomycin B treatment. We introduced point mutations within the K17-NES (mNES) and the K17-NLS (mNLS). Nuclear p27KIP1 was lost in cells expressing wild-type K17. In contrast, nuclear p27KIP1 levels were 3-fold higher in cells expressing either mNLS or mNES. Furthermore, nuclear localization of K17 was abolished in mNLS cells. Xenografts of cervical cancer cells showed that tumors derived from K17 expressing cells were 3-times larger than those derived from K17 knockdown cells, the latter showing increased nuclear p27KIP1 and decreased PCNA and Ki67 expression. Finally, we identified an inverse correlation between K17 and p27KIP1 expression in human cervical cancer specimens, intertumorally and intratumorally. These data suggest a model in which nuclear-localized K17 acts as an oncoprotein promoting G1/S transition in cancer cells, by mediating exportin binding and nuclear translocation of tumor suppressor p27KIP1, contributing to sustained proliferation, tumor aggressiveness and poor-patient outcome. Citation Format: Luisa F. Escobar-Hoyos, Ruchi Shah, Lucia Roa-Peña, Nilofar Najafian, Elizabeth Vanner, Anna Banach, Erik Nielsen, Ramsey Al-Khalil, Ali Akalin, David Talmage, Kenneth Shroyer. Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2015-2036

Abstract 2442: XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing

Abstract Human Exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports a variety of different cargo proteins out of the cell's nucleus. Inhibition of XPO1-mediated protein export results in nuclear retention of cargo proteins including tumour suppressor proteins and is a novel therapeutic strategy for cancer. Selinexor, an orally bioavailable XPO1 inhibitor, effectively demonstrates potent activity against multiple types of cancer. Here we directly demonstrate the high selectivity of selinexor for XPO1 inside living cells. Selinexor binds to the cargo-binding pocket of XPO1 and inhibits the formation of XPO1-cargo complex. The binding of selinexor to XPO1 involves a slowly reversible Michael addition type interaction of its acrylate moiety with cysteine 528 residue of XPO1. Using CRISPR/Cas9 genome editing we have now created a homozygous Jurkat T-ALL cell line carrying a Cys528Ser mutation in XPO1. This cell line was used to study the specificity of selinexor for XPO1 inside the cancer cell. Selinexor was highly toxic to wild-type Jurkat cells in the low nanomolar range while the mutant cells were resistant up to micromolar concentrations. These results were confirmed by resistance to induction of caspase dependent apoptosis as well as cell cycle arrest in mutant cells. The single Cys528Ser mutation thus confers resistance to selinexor. In addition, selinexor failed to accumulate cargo proteins in the nucleus of mutant cells. Moreover, the Cys528Ser substitution conferred resistance to the direct interaction of this drug to XPO1 in cells. In conclusion, these results validate XPO1 as the selinexor's prime target and pinpoint its unique selectivity to the Cys528 residue of XPO1. Our findings also demonstrate the general utility of CRISPR/Cas9 genome editing for introducing point mutations in the genome of cancer cells for drug-target specificity validation. Citation Format: Jasper E. Neggers, Thomas Vercruysse, Maarten Jacquemyn, Els Vanstreels, Erkan Baloglu, Sharon Shacham, Marsha Crochiere, William Senapedis, Yosef Landesman, Dirk Daelemans. XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2015-2442

Pex11mediates peroxisomal proliferation by promoting deformation of the lipid membrane.

Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pβ caused substantial vesiculation of peroxisomes in mammalian cells. This vesicle formation was dependent on dynamin-like protein 1 (DLP1) and mitochondrial fission factor (Mff), as knockdown of these proteins diminished peroxisomal fission after Pex11pβ overexpression. The fission-deficient peroxisomes exhibited an elongated morphology, and peroxisomal marker proteins, such as Pex14p or matrix proteins harboring peroxisomal targeting signal 1, were discernible in a segmented staining pattern, like beads on a string. Endogenous Pex11pβ was also distributed a striped pattern, but which was not coincide with Pex14p and PTS1 matrix proteins. Altered morphology of the lipid membrane was observed when recombinant Pex11p proteins were introduced into proteo-liposomes. Constriction of proteo-liposomes was observed under confocal microscopy and electron microscopy, and the reconstituted Pex11pβ protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pβ strongly reduced peroxisomal fission, and decreased the oligomer formation. These results suggest that Pex11p contributes to the morphogenesis of the peroxisomal membrane, which is required for subsequent fission by DLP1.

Structural studies and cytotoxicity assays of "aggregation-prone" IAPP(8-16) and its non-amyloidogenic variants suggest its important role in fibrillogenesis and cytotoxicity of human amylin.

Amyloid deposits to the islets of Langerhans are responsible for the gradual loss of pancreatic β-cells leading to type II diabetes mellitus. Human mature islet amyloid polypeptide (hIAPP), a 37-residue pancreatic hormone, has been identified as the primary component of amyloid fibrils forming these deposits. Several individual segments along the entire sequence length of hIAPP have been nominated as regions with increased amyloidogenic potential, such as regions 8-20, 20-29, and 30-37. A smaller fragment of the 8-20 region, spanning residues 8-16 of hIAPP has been associated with the formation of early transient α-helical dimers that promote fibrillogenesis and also as a core part of hIAPP amyloid fibrils. Utilizing our aggregation propensity prediction tools AmylPred and AmylPred2, we have identified the high aggregation propensity of the 8-16 segment of hIAPP. A peptide analog corresponding to this segment was chemically synthesized and its amyloidogenic properties were validated using electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, and polarized microscopy. Additionally, two peptides introducing point mutations L12R and L12P, respectively, to the 8-16 segment, were chemically synthesized. Both mutations disrupt the α-helical properties of the 8-16 region and lower its amyloidogenic potential, which was confirmed experimentally. Finally, cytotoxicity assays indicate that the 8-16 segment of hIAPP shows enhanced cytotoxicity, which is relieved by the L12R mutation but not by the L12P mutation. Our results indicate that the chameleon properties and the high aggregation propensity of the 8-16 region may significantly contribute to the formation of amyloid fibrils and the overall cytotoxic effect of hIAPP.

Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site.

Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride's positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound's potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

Immuno-evasive tactics by schistosomes identify an effective allergy preventative

Many chronic inflammatory diseases can be improved by helminth infection, but the mechanisms are poorly understood. Allergy and helminthiasis are both associated with Th2-like immune responses; thus, defining how infection with parasites leads to reduced allergy has been particularly challenging. We sought to better understand this conundrum by evaluating host–parasite interactions involved in Th2 immunity in human schistosomiasis. Immune cells were cultured with schistosomes and the effect on CD23, an IgE receptor associated with resistance in schistosomiasis, was evaluated. Cells treated with schistosomes demonstrated reduced surface CD23 levels with a parallel accumulation of soluble (s) CD23 suggesting this IgE receptor is proteolytically cleaved by the parasite. Consistent with this hypothesis, a schistosome-generated (SG)-sCD23 fragment of 15 kDa was identified. SG-sCD23 inhibited IgE from binding to CD23 and FcεRI, but lacked the ability to bind CD21. These results suggested that schistosomes target IgE-mediated immunity in immuno-evasive tactics. Based on its characteristics, we predicted that SG-sCD23 would function as an efficacious allergy preventative. Treatment of human FcεRI-transgenic mice with recombinant (r) SG-sCD23 reduced the ability of human IgE to induce an acute allergic response in vivo. In addition, an optimized form of rSG-sCD23 with an introduced point mutation at Asp258 (D258E)to stabilize IgE binding had increased efficacy compared to native rSG-sCD23. Schistosome infection may thus inhibit allergic-like protective immune responses by increasing soluble IgE decoy receptors. Allergy treatments based on this naturally occurring phenomenon may be highly effective and have fewer side effects with long-term use.

Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity

Prokaryotic FAD synthetases (FADSs) are bifunctional enzymes composed of two modules, the C-terminal module with ATP:riboflavin kinase (RFK) activity, and the N-terminus with ATP:FMN adenylyltransferase (FMNAT) activity. The FADS from Corynebacterium ammoniagenes, CaFADS, forms transient oligomers during catalysis. These oligomers are stabilized by several interactions between the RFK and FMNAT sites from neighboring protomers, which otherwise are separated in the monomeric enzyme. Among these inter-protomer interactions, the salt bridge between E268 at the RFK site and R66 at the FMNAT-module is particularly relevant, as E268 is the catalytic base of the kinase reaction. Here we have introduced point mutations at R66 to analyze the impact of the salt-bridge on ligand binding and catalysis. Interestingly, these mutations have only mild effects on ligand binding and kinetic properties of the FMNAT-module (where R66 is located), but considerably impair the RFK activity turnover. Substitutions of R66 also modulate the ratio between monomeric and oligomeric species and modify the quaternary arrangement observed by single-molecule methods. Therefore, our data further support the cross-talk between the RFK- and FMNAT-modules of neighboring protomers in the CaFADS enzyme, and establish the participation of R66 in the modulation of the geometry of the RFK active site during catalysis.