Linker Sequence Research Articles

Enhanced anti-cancer effect using MMP-responsive L-asparaginase fused with cell-penetrating 30Kc19 protein

As the acute lymphoblastic leukaemia (ALL) develops, expression of L-asparaginase (ASNase) protein is known to decrease. Therefore, deficiency of the ASNase protein would be regarded as one of the significant indications of the ALL. For the treatment of ALL, recombinant ASNase protein derived from bacterial origin is used which causes cytotoxicity by deprivation of Asn. However, short half-life of the protein is an obstacle for medical use. In order to overcome this limit, recombinant ASNase was fused to 30Kc19 with protein-stabilizing and cell-penetrating properties. As the 30Kc19 protein may induce steric hindrance, we further added a PLGLAG linker sequence (LK) between the ASNase and 30Kc19. The treatment of ASNase-LK-30Kc19 fusion protein demonstrated enhanced stability, cell-penetrating property, and anti-cancer activity. Intracellular delivery of both the non-cleaved and cleaved forms of the protein were observed, suggesting that ASNase acted both internally and externally, performing high anti-cancer activity by effective depletion of intracellular Asn. Additionally, ASNase-LK-30Kc19 showed high selectivity towards cancer cells. In terms of the dosage, releasable ASNase from ASNase-LK-30Kc19 reached the same half-maximal inhibitory concentration at a concentration five times lower than non-releasable ASNase-30Kc19. Altogether, the findings suggest that this fusion approach has potential applications in the treatment of ALL.

Precursor genes of Bowman-Birk-type serine proteinase inhibitors comprise multiple inhibitory domains to promote diversity

BackgroundProteinase inhibitors are important for the regulation of the activity of enzymes essential for the survival and maintenance of all organisms, and they may hold medicinal and agricultural value. Hyacinthus orientalis L. serine protease inhibitors (HOSPIs), belonging to the Bowman–Birk type inhibitor (BBI) family, have strong inhibitory activities against mammalian serine proteinases. This study explored the relationship between gene structure and multiple isoinhibitor production of these diversified BBIs by analyzing sequences of HOSPI precursor genes. MethodsGenomic DNA of H. orientalis roots was obtained and fragmented using 13 specific restriction enzymes, which were amplified by inverse and nested polymerase chain reactions, cloned into the pBluescript II SK (+) vector, and directly sequenced using specific primers. HOSPI gene and protein expression were assessed by quantitative real-time PCR and western blot, respectively. Proteinase inhibitory activity of hyacinth bulb extracts was evaluated by fluorescein isothiocyanate-labeled casein. ResultsFour distinct HOSPI precursor genes were identified, encoding 2–4 different HOSPI domains that were surrounded by additional sequences (named head, linker, and tail sequences) and some introns. Moreover, 3′ splicing of the linker sequence may occur through introns inserted between linker sequences. HOSPI gene and protein expression was higher during the stem elongation and the flowering periods. ConclusionsThese results indicate that gene duplication of the HOSPI precursor as a single set, including tandem repeated HOSPI domains, leads to diversity and effective production of mature HOSPIs by posttranslational processing. General significanceThese findings shed light on the diversity of proteinase inhibitors.

Engineering an Fc-Fusion of a Capsule Degrading Enzyme for the Treatment of Anthrax.

Polymers of d-glutamic acid (PDGA) form the capsule of the highly virulent Ames strain of B.anthracis. PDGA is antiphagocytic and weakly immunogenic; it enables the bacteria to evade the innate immune responses. CapD is an enzyme that catalyzes the covalent anchoring of PDGA. CapD is an Ntn-amido hydrolase that utilizes an internal Thr-352 as its nucleophile and general acid and base. An internal cleavage produces a free N-terminal Thr-352 and a short and long polypeptide chain. The chains were circularly permuted (CP) to move Thr-352 to the N-terminus of the polypeptide. We previously showed that a branched PEG-CapDS334C-CP could protect mice (80% survival) against a 5 LD50 challenge with B.anthracis Ames without the use of antibiotics, monoclonals, or vaccines. In attempts to improve the in vivo circulation time of CapD and enhance its avidity to its polymeric substrate, an Fc-domain of a mouse IgG1 was fused to CapDS334C-CP and the linker length and sequence were optimized. The resulting construct, Fc-CapDS334C-CP, then was pegylated with a linear 2 kDa mPEG at S334C to produce mPEG-Fc-CapDS334C-CP. Interestingly, the fusion of the Fc-domain and incorporation of the S334C mutation imparted acid stability, but slightly reduced the kcat (∼ 2-fold lower). In vivo, the measured protein concentration in sera was higher for the Fc-fusion constructs compared to the mPEG-Fc-CapDS334C-CP. However, the exposure calculated from measured sera enzymatic activity was higher for the mPEG-CapDS334C-CP. The pegylated Fc-fusion was less active than the PEG-CapDS334C-CP, but detectable in sera at 24 h by immunoblot. Here we describe the engineering of a soluble, active, pegylated Fc-fusion of B.anthracis CapD (mPEG-Fc-CapD-CP) with activity in vitro, in serum, and on encapsulated bacteria.

Enzymatic Formation of Recombinant Antibody-Conjugated Gold Nanoparticles in the Presence of Citrate Groups and Bacteria

With the spread of deadly diseases worldwide, the design of rapid tests to identify causative microorganisms is necessary. Due to the unique properties of gold nanoparticles, these nanoparticles are used in designing rapid diagnostic tests, such as strip tests. The current study aimed to investigate the ability of gold nanoparticles to bind to single-chain variable fragment antibodies. In this study, the biological and chemical methods included Escherichia coli TOP-10 and the Turkevich method to synthesize the gold nanoparticles, respectively. Then, the effect of synthetic nanoparticles on their capability of binding to recombinant antibodies was assessed by agarose gel and UV-vis spectroscopy. Our result showed that gold nanoparticles had a spherical morphology, and their average size was ~45 nm. Additionally, the citrate groups in gold nanoparticles were able to bind to serine residues in the antibody linker sequence; so, the chemical synthesis of gold nanoparticles is an effective strategy for binding these nanoparticles to antibodies that can be used in designing rapid diagnostic tests to promptly identify infectious microorganisms.

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti-HpTGEKP Antibody and Zinc Finger Protein Linker Phospho-Hexapeptides.

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were C-terminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.

Calmodulin's Interdomain Linker Is Optimized for Dynamics Signal Transmission and Calcium Binding.

Linkers are ubiquitous in multidomain proteins. These linkers are integral to protein functions, and accumulating evidence suggests that the linkers' versatile roles are encoded in their sequences. However, a molecular picture of how amino acid differences in the linker influence protein function is still lacking. By using extensive Gaussian-accelerated MD coupled with dynamic network analysis, we reveal the molecular bases underlying the linker's role in Calmodulin (CaM), a highly conserved Ca2+-signaling hub in eukaryotes. Three CaM constructs comprising a wild-type linker, a flexible linker (four glycines at position D78-S81), and a rigid linker (four prolines at position D78-S81) were simulated. We show that the flexible linker resembles the wild type in allowing CaM to sample a large ensemble of conformations while the rigid linker confines the sampling. Our simulations recapture experimental observations that target binding enhances the Ca2+ affinity to CaM's EF-hand sites at the N-domain. However, only the wild-type linker can both correctly capture the Ca2+ binding order and maintain the α-helical structure of the domain. The other two constructs either bind Ca2+ in an incorrect order or exhibit unfolding of an N-domain helix. We demonstrate that the wild-type linker achieves these outcomes by transmitting interdomain dynamics efficiently. This was evidenced by stronger (anti)correlations among the linker residues, decoupling of the hydrogen bonds between A1-A15 and V35-E45, and structuring of the N-domain for Ca2+ binding. This decoupling was not evident for the other two constructs. Lastly, we show that the wild-type linker's optimal transmission stems from its thermodynamically favorable strain and solvation relative to the other two constructs. Our results show how the linker sequence tunes CaM function, suggesting possible mechanisms for changes in linker properties such as mutations or post-translational modifications to modulate protein/substrate binding.

Portable and sensitive detection of miRNA in cerebral infarction through the DSN enzyme assisted dual signal recycles by using personal glucose meters (PGMs)

Development of a portable and sensitive microRNAs (miRNAs) detection approach is crucial for the fundamental research and early-diagnosis of many diseases. Herein, we propose a novel miRNA detection strategy by using duplex-specfic nuclease (DSN enzyme) and DNAzyme to form dual signal amplification, and using PGMs to read the detection result. In this method, a hairpin structure catch probe is designed to specifically recognize target miRNA to generate a DNA-RNA complex. Under the assistance of DSN enzyme, the binding section which is complementary with target miRNA is digested, and the miRNA is released from the duplex. The liberated miRNA unfolds a next catch probe to form the first signal amplification. Meanwhile, the DNAzyme section in catch probe forms its active structure under the assistance of Mg2+ and substrate to cut the complementary sequence in substrate and to generate the linker. The linker sequence assists the formation of SMBs-ssDNA-sucrase complex through mediating conjugation of sucrase-ssDNA and streptavidin magnetic beads (SMBs)-ssDNA, generating. After magnet based removal of unbound linker sequences, the complex is then utilized to catalyze sucrose to glucose, which could be detected by using PGMs. Based on this, the approach exhibits a favorable detection sensitivity with a low limit of detection (LOD). This new approach is an essential step toward the portable and reliable detection of miRNA for diagnostic purposes.

Abstract 584: Driving anti-tumor activity in solid tumors with controlled arming of allogeneic CAR-NK cells

Abstract Recent successes with cell therapies, including CAR-T and CAR-NK cells, for hematopoietic cancers have yet to translate into solid tumors. The major obstacle solid tumors present for cell therapy is the highly immunosuppressive tumor microenvironment (TME), which can impair the function of endogenous as well as therapeutic immune cells. To overcome this challenge, Senti Bio is developing gene circuits to arm CAR-NK cells with multiple cytokines to improve the cytotoxicity and persistence of CAR-NK product candidates. Cytokines, as well as other immune effectors, can act in autocrine or paracrine manner, benefiting the CAR-NK cells as well as potentially engaging the local immune system in the TME. Secreted cytokines can be widely distributed and stimulate other immune cells in the TME, however this is often associated with systemic toxicity as well as reduced impact on the CAR-NK cells. On the other hand, membrane-bound cytokines result in a localized and potent stimulation of the CAR-NK cells but have limited impact on the broader TME. Since IL-15 is known to stimulate NK cell survival and function, Senti Bio has created a novel calibrated release (cr) technology to specifically optimize IL-15 signaling in its CAR-NK platform. crIL-15 is tethered to the NK cells via a cleavable linker that allows for IL-15 release upon cleavage by a ubiquitously expressed protease on the cell surface of NK cells. The rate of linker cleavage can be calibrated by engineering the sensitivity of the cleavable linker sequence to the cell surface protease, thereby enabling the tuning of the ratio of membrane tethered versus fully secreted protein. We compared the function of the wildtype IL-15 protein with different variants of crIL-15 that have a faster or slower release. We demonstrated that crIL-15 has a more potent function, stimulating pSTAT5 signaling and driving expansion and survival of NK cells. CAR-NK cells armed with crIL-15 significantly outperformed CAR-NK cells that expressed the wildtype IL-15 protein, had enhanced function, and demonstrated significantly improved cytotoxicity over three rounds of serial killing (p<0.0001). In parallel, we armed CAR-NK cells with gene circuits that encoded various combinations of two or more cytokines. In particular, the combination of IL-15 and IL-21 resulted in a synergistic effect, leading to longer survival in the absence of cytokines compared to NK cells armed with IL-15 alone. Furthermore, CAR-NK cells armed with IL-15 + IL-21 had significantly increased activity and cytotoxicity compared to CAR-NK cells armed with either cytokine alone (up to 4-times increased cytotoxicity, p<0.0001) when co-cultured with tumor cells. Overall, our results suggest the advantage of multi-arming CAR-NK cells with cytokines, using controlled release systems and cytokine combinations, resulting in improved NK cell function in solid tumor models. Citation Format: Alba Gonzalez, Michelle Hung, Marcela Guzman, Aldo Sotelo, Nicholas Frankel, Yin Yin Chong, Deepika Kaveri, Poornima Ramkumar, Elizabeth Leitner, Priscilla Wong, Ronni Ponek, Kelly Lee, Alyssa L. Mullenix, Russell M. Gordley, Gary Lee. Driving anti-tumor activity in solid tumors with controlled arming of allogeneic CAR-NK cells [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 584.

Combination of chemotherapy and immune checkpoint therapy by the immunoconjugates-based nanocomplexes synergistically improves therapeutic efficacy in SCLC

Although the etoposide and carboplatin (EP) combination strategy has been the first-line chemotherapy, patients with extensive-stage disease small-cell lung cancer (SCLC) still have poor survival outcomes. Our retrospective analysis revealed that 46 patients with SCLC only achieved medium overall survival (OS) of 11.6 months after treated by EP. Recently, it was demonstrated that combination therapy of PD1/PD-L1 immune checkpoint blocker and EP could significantly improve the OS of SCLC patients. However, the serious treatment-related toxicity leaded to a high rate of treatment-discontinuation or even death. In the present study, we have developed a novel TPP1-conjugated nanocomplex, abbreviated as TPP1NP-EP, which was co-loaded with carboplatin (CBP) and etoposide (VP16). The TPP1 was a PD-L1 targeting peptide and conjugated on the surface of nanocomplex by a matrix metalloproteinase (MMP-2/9)-cleavable peptide linker sequence PLGLAG. For dual-loading of CBP and VP16, the CBP was chemically conjugated with poly(ethylene glycol) (PEG)–poly(caprolactone) (PCL) by pH-sensitive hydrazone bond and the VP16 was physically encapsulated by emulsion-solvent evaporation method. In vitro and in vivo experiments demonstrated an excellent anti-tumor effect of TPP1NP-EP on SCLC and improved safety. In conclusion, the present study has provided a promising strategy for treatment of malignant SCLC.

Identification of vaccine targets & design of vaccine against SARS-CoV-2 coronavirus using computational and deep learning-based approaches.

An unusual pneumonia infection, named COVID-19, was reported on December 2019 in China. It was reported to be caused by a novel coronavirus which has infected approximately 220 million people worldwide with a death toll of 4.5 million as of September 2021. This study is focused on finding potential vaccine candidates and designing an in-silico subunit multi-epitope vaccine candidates using a unique computational pipeline, integrating reverse vaccinology, molecular docking and simulation methods. A protein named spike protein of SARS-CoV-2 with the GenBank ID QHD43416.1 was shortlisted as a potential vaccine candidate and was examined for presence of B-cell and T-cell epitopes. We also investigated antigenicity and interaction with distinct polymorphic alleles of the epitopes. High ranking epitopes such as DLCFTNVY (B cell epitope), KIADYNKL (MHC Class-I) and VKNKCVNFN (MHC class-II) were shortlisted for subsequent analysis. Digestion analysis verified the safety and stability of the shortlisted peptides. Docking study reported a strong binding of proposed peptides with HLA-A*02 and HLA-B7 alleles. We used standard methods to construct vaccine model and this construct was evaluated further for its antigenicity, physicochemical properties, 2D and 3D structure prediction and validation. Further, molecular docking followed by molecular dynamics simulation was performed to evaluate the binding affinity and stability of TLR-4 and vaccine complex. Finally, the vaccine construct was reverse transcribed and adapted for E. coli strain K 12 prior to the insertion within the pET-28-a (+) vector for determining translational and microbial expression followed by conservancy analysis. Also, six multi-epitope subunit vaccines were constructed using different strategies containing immunogenic epitopes, appropriate adjuvants and linker sequences. We propose that our vaccine constructs can be used for downstream investigations using in-vitro and in-vivo studies to design effective and safe vaccine against different strains of COVID-19.

The Chimeric Adenovirus (Ad5/35) Expressing Engineered Spike Protein Confers Immunity against SARS-CoV-2 in Mice and Non-Human Primates.

Several COVID-19 platforms have been licensed across the world thus far, but vaccine platform research that can lead to effective antigen delivery is still ongoing. Here, we constructed AdCLD-CoV19 that could modulate humoral immunity by harboring SARS-CoV-2 antigens onto a chimeric adenovirus 5/35 platform that was effective in cellular immunity. By replacing the S1/S2 furin cleavage sequence of the SARS-CoV-2 Spike (S) protein mounted on AdCLD-CoV19 with the linker sequence, high antigen expression was confirmed in various cell lines. The high levels of antigen expression contributed to antigen-specific antibody activity in mice and non-human primates (NHPs) with a single vaccination of AdCLD-CoV19. Furthermore, the adenovirus-induced Th1 immune response was specifically raised for the S protein, and these immune responses protected the NHP against live viruses. While AdCLD-CoV19 maintained neutralizing antibody activity against various SARS-CoV-2 variants, it was reduced to single vaccination for β and ο variants, and the reduced neutralizing antibody activity was restored with booster shots. Hence, AdCLD-CoV19 can prevent SARS-CoV-2 with a single vaccination, and the new vaccine administration strategy that responds to various variants can maintain the efficacy of the vaccine.

Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating.

Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products with potent antibiotic, antiviral, and anticancer properties. RiPP enzymes known as cyclodehydratases and dehydrogenases work together to catalyze intramolecular, inter-residue condensation and dehydrogenation reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here, we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomers—including aminobenzoic acid derivatives and beta-amino acids—within leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse non-α-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bond—both features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

SAC-TRAIL, a novel anticancer fusion protein: expression, purification, and functional characterization.

Recombinant protein pharmaceutical agents have been widely used for cancer treatment. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has broad-spectrum antitumor activity, its clinical applications are limited because most tumor cells eventually develop resistance to TRAIL-induced apoptosis through various pathways. Prostate apoptosis response-4 (Par-4) selectively induces apoptosis in cancer cells after binding to the cell surface receptor, GRP78. In this study, TRAIL was fused with the core domain of Par-4 (SAC) to produce a novel recombinant fusion protein. To obtain solubly expressed fusion protein, a small ubiquitin-related modifier (SUMO) was added to the N-terminus of the target protein. Cytotoxicity assays showed that the purified fusion protein exhibited more significant antitumor activity on cancer cells than that by native TRAIL. The connection order and linker sequence of the fusion proteins were optimized. In vitro cytotoxicity assay showed that the SAC-TRAIL fusion protein, which contained a flexible linker (G4S)3, optimally inhibited the proliferation of cancer cells. Immunofluorescence assays demonstrated that SAC-TRAIL could efficiently and specifically bind to cancer cells. Additionally, circular dichroism assays showed that the secondary structure of the recombinant protein with a flexible linker (G4S)3 has both a lower α-helix and higher random coiling, which facilitates the specific binding of SAC-TRAIL to the receptor. Collectively, these results suggest that the novel recombinant fusion protein SAC-(G4S)3-TRAIL is a potential therapeutic agent for cancer. KEY POINTS: • Improved tumor growth suppression and apoptosis induction potency of SAC-TRAIL. • Enhanced targeting selectivity of SAC-TRAIL in cancer cells. • Lower α-helix and higher random coiling in SAC-TRAIL with flexible linker (G4S)3.

Evolutionary analyses of the gasdermin family suggest conserved roles in infection response despite loss of pore-forming functionality

BackgroundGasdermins are ancient (>500million-years-ago) proteins, constituting a family of pore-forming proteins that allow the release of intracellular content including proinflammatory cytokines. Despite their importance in the immune response, and although gasdermin and gasdermin-like genes have been identified across a wide range of animal and non-animal species, there is limited information about the evolutionary history of the gasdermin family, and their functional roles after infection.In this study, we assess the lytic functions of different gasdermins across Metazoa species, and use a mouse model of sepsis to evaluate the expression of the different gasdermins during infection.ResultsWe show that the majority of gasdermin family members from distantly related animal clades are pore-forming, in line with the function of the ancestral proto-gasdermin and gasdermin-like proteins of Bacteria. We demonstrate the first expansion of this family occurred through a duplication of the ancestral gasdermin gene which formed gasdermin E and pejvakin prior to the divergence of cartilaginous fish and bony fish ~475 mya. We show that pejvakin from cartilaginous fish and mammals lost the pore-forming functionality and thus its role in cell lysis. We describe that the pore-forming gasdermin A formed ~320 mya as a duplication of gasdermin E prior to the divergence of the Sauropsida clade (the ancestral lineage of reptiles, turtles, and birds) and the Synapsid clade (the ancestral lineage of mammals). We then demonstrate that the gasdermin A gene duplicated to form the rest of the gasdermin family including gasdermins B, C, and D: pore-forming proteins that present a high variation of the exons in the linker sequence, which in turn allows for diverse activation pathways. Finally, we describe expression of murine gasdermin family members in different tissues in a mouse sepsis model, indicating function during infection response.ConclusionsIn this study we explored the evolutionary history of the gasdermin proteins in animals and demonstrated that the pore-formation functionality has been conserved from the ancient proto-gasdermin protein. We also showed that one gasdermin family member, pejvakin, lost its pore-forming functionality, but that all gasdermin family members, including pejvakin, likely retained a role in inflammation and the physiological response to infection.

Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence.

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.

Development of a novel liquid crystal Apta-sensing platform using P-shape molecular switch

Described herein is a liquid crystal (LC)-based aptasensor via employing the reorientation of LC triggered by the conformational changes of a P-shaped DNA structure. The structure consists of a short linker sequence as an immobilizer probe with ability to hybridize with the central part of the intact aptamer (Apt) sequence and an Apt terminal-locker (ATL) strand with complementary segments of the Apt terminal fragments. Bindings of two arm segments of the Apt sequence with the ATL strand enforces it to form a P-shaped configuration on the sensing platform. The selective interaction between the Apt strand and OTA leads to the disassembly of the Apt-ATL hybrid, collapse of the P-shaped structure, and consequently, transition of the optical appearance of the aptasensor texture. Determination of Ochratoxin A (OTA) in foods is an urgent demand in attempt to minimize food safety risks. To demonstrate the feasibility of our aptasensing design, the OTA specific aptamer was selected as a model. The developed LC aptasensor possesses a wide linear range from 0.01 aM to 100 pM, ultra-low limit of detection (LOD) of 0.0078 aM, and quantitative recoveries of 91–103.51% for OTA in rice and grape juice samples. This study proposes a novel and universal LC-based platform for facile, ultra-sensitive, and precision sensing of hazardous analytes in real samples.

Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralization

Mineralization catalyzed by carbonic anhydrase (CA) is one of the most promising technologies for capturing CO2. In this work, Escherichia coli BL21(DE3) was used as the host, and the N-terminus of ice nucleation protein (INPN) was used as the carrier protein. Different fusion patterns and vectors were used to construct CA surface display systems for α-carbonic anhydrase (HPCA) from Helicobacter pylori 26695 and α-carbonic anhydrase (SazCA) from Sulfurihydrogenibium azorense. The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity, while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system. The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA. Cell fractionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell's outer membrane as a fusion protein. The enzyme activity of the surface display strain E-22b-IRLS (11.43 U·mL−1OD600−1) was significantly higher than that of the intracellular expression strain E-22b-S (8.355 U·mL−1OD600−1) under optimized induction conditions. Compared with free SazCA, E-22b-IRLS had higher thermal and pH stability. The long-term stability of SazCA was also significantly improved by surface display. When the engineered strain and free enzyme were used for CO2 mineralization, the amount of CaCO3 deposition catalyzed by the strain E-22b-IRLS on the surface (241 mg) was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S (173 mg). These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO2 capture and mineralization.

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis.

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the linker sequence for the mature LASSO probe through Cre-LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (≈46% of correct probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 Escherichia coli ORFs spanning from 400- to 5000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, were able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.