Mutation Of Residues Research Articles

Development of an Engineered Sugar Aminotransferase with Simultaneously Improved Stability and Non-Natural Substrate Activity to Synthesize the Glucosidase Inhibitor Valienamine

Sugar aminotransferases (SATs) catalyze the installation of chiral amines onto specific keto sugars, producing bioactive amino sugars. Their activity has been utilized in artificial reactions, such as using the SAT WecE to transform valienone into the valuable α-glucosidase inhibitor valienamine. However, the low thermostability and limited activity on non-natural substrates have hindered their applications. Simultaneously improving stability and enzyme activity is particularly challenging owing to the acknowledged inherent trade-off between stability and activity. A customized combinatorial active-site saturation test-iterative saturation mutagenesis (CAST-ISM) strategy was used to simultaneously enhance the stability and activity of WecE toward valienone. Fourteen hotspots related to improving the stability–\\activity trade-off were identified based on evolutionary conservation and the average mutation folding energy assessment of 57 residues in the active site of WecE. Positive mutagenesis and combinatorial mutations of these specific residues were accomplished via site-directed saturation mutagenesis (SSM) and iterative evolution cycles. Compared with those of the wild-type (WT) WecE, the quadruple mutant M4 (Y321F/K209F/V318R/ F319V) displayed a 641.49-fold increase in half-life (t1/2) at 40 °C and a 31.37-fold increase in activity toward the non-natural substrate valienone. The triple mutant M3 (Y321F/K209F/V318R) demonstrated an 83.04-fold increase in (t1/2) at 40 °C and a 37.77-fold increase in activity toward valienone. The underlying mechanism was dependent on the strengthened interface interactions and shortened transamination reaction catalytic distance, compared with those of the WT, which improved the stability and activity of the obtained mutants. Thus, we accomplished a general target-oriented strategy for obtaining stable and highly active SATs for artificial amino-sugar biosynthesis applications.

The oncogenic fusion protein EML4-NTRK3 requires three salt bridges for stability and biological activity

Aim of studyChromosomal translocations involving neurotrophic receptor tyrosine kinases (NTRKs) have been identified in 20 % of soft tissue sarcomas. This work focuses on the EML4-NTRK3 translocation identified in cases of Infantile Fibrosarcoma, which contains the coiled-coil multimerization domain of Echinoderm Microtubule-like protein 4 (EML4) fused with the tyrosine kinase domain of Neurotrophic Receptor Tyrosine Kinase 3 (NTRK3). The aim of the study was to test the importance of tyrosine kinase activity and multimerization for the oncogenic activity of EML4-NTRK3. MethodsThese studies examined EML4-NTRK3 proteins containing a kinase-dead or WT kinase domain, together with mutations in specific salt bridge residues within the coiled-coil domain. Biological activity was assayed using focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT pathways were analyzed for downstream activation of signaling pathways. Localization of EML4-NTRK3 proteins was examined by immunofluorescence microscopy, and the ability of the EML4 coiled-coil domain to drive protein multimerization was examined by biochemical assays. ResultsActivation of EML4-NTRK3 relies on both the tyrosine kinase activity of NTRK3 and salt-bridge stabilization within the coiled-coil domain of EML4. The tyrosine kinase activity of NTRK3 is essential for the biological activation of EML4-NTRK3. Furthermore, EML4-NTRK3 activates downstream signaling pathways MAPK/ERK, JAK/STAT3 and PKC/PLCγ. The disruption of three specific salt bridge interactions within the EML4 coiled-coil domain of EML4-NTRK3 blocks downstream activation, biological activity, and the ability to hetero-multimerize with EML4. We also demonstrate that EML4-NTRK3 is localized in the cytoplasm and fails to associate with microtubules. Concluding statementThese data suggest potential therapeutic strategies for Infantile Fibrosarcoma cases bearing EML4-NTRK3 fusion through inhibition of salt bridge interactions and disruption of multimerization.

Functional roles of the interaction of Moa1 with CENP-C and Rec8 in meiosis of Schizosaccharomyces pombe.

The localization of the meiotic specific regulatory molecule Moa1 to the centromere is regulated by the kinetochore protein CENP-C, and participates in the cohesion of sister chromatids in the centromere region mediated by the cohesin Rec8. To examine the interaction of these proteins, we analyzed the interactions between Moa1 and Rec8, CENP-C by yeast two-hybrid assays and identified several amino acid residues in Moa1 required for the interaction with CENP-C and Rec8. The results revealed that the interaction between Moa1 and CENP-C is crucial for the Moa1 to participate in the regulation of monopolar attachment of sister kinetochores. However, mutation at S143 and T150 of Moa1, which are required for interaction with Rec8 in the two-hybrid assay, did not show significant defects. Mutations in amino acid residues may not be sufficient to interfere with the interaction between Moa1 and Rec8 in vivo. Further research is needed to determine the interaction domain between Moa1 and Rec8. This study revealed specific amino acid sites at which Moa1 affects the meiotic homologous chromosome segregation, providing a deeper understanding of the mechanism of meiotic chromosome segregation.

Structural and functional characterization of the nucleotide-binding domains of ABCA4 and their role in Stargardt disease

ABCA4 is an ATP-binding cassette (ABC) transporter that prevents the buildup of toxic retinoid compounds by facilitating the transport of N-retinylidene-phosphatidylethanolamine across membranes of rod and cone photoreceptor cells. Over 1500 missense mutations in ABCA4, many in the nucleotide binding domains (NBDs), have been genetically linked to Stargardt disease (STGD1). Here, we show by Cryo-electron microscopy that ABCA4 is converted from an open outward conformation to a closed conformation upon the binding of AMP-PNP. Structural information and biochemical studies were used to further define the role of the NBDs in the functional properties of ABCA4 and the mechanisms by which mutations lead to the loss in activity. We show that ATPase activity in both NBDs is required for the functional activity of ABCA4. Mutations in Walker A asparagine residues cause a severe reduction in substrate-activated ATPase activity due to the loss in polar interactions with residues within the D-loops of the opposing NBD. The structural basis for how disease mutations in other NBD residues including the R1108C, R2077W, R2107H and L2027F affect the structure and function of ABCA4 is described. Collectively, our studies provide insight into the structure and function of ABCA4 and mechanisms underlying STGD1.

Molecular insight into the neutralization mechanism of human-origin monoclonal antibody AH100 against Hantaan virus.

Both Old World and New World hantaviruses are transmitted through rodents and can lead to hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome in humans without the availability of specific therapeutics. The square-shaped surface spikes of hantaviruses consist of four Gn-Gc heterodimers that are pivotal for viral entry into host cells and serve as targets for the immune system. Previously, a human-derived neutralizing monoclonal antibody, AH100, demonstrated specific neutralization against the Old World hantavirus, Hantaan virus. However, the precise mode binding of this neutralizing monoclonal antibody remains unclear. In the present study, we determined the structure of the Hantaan virus Gn-AH100 antigen-binding fragment complex and identified its epitope. Crystallography revealed that AH100 targeted the epitopes on domain A and b-ribbon and E3-like domain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike revealed its localization between neighboring Gn protomers, distinguishing this epitope as a unique site compared to the previously reported monoclonal antibodies. This study provides crucial insights into the structural basis of hantavirus neutralizing antibody epitopes, thereby facilitating the development of therapeutic antibodies.IMPORTANCEHantaan virus (HTNV) poses a significant threat to humans by causing hemorrhagic fever with renal syndrome with high mortality rates. In the absence of FDA-approved drugs or vaccines, it is urgent to develop specific therapeutics. Here, we elucidated the epitope of a human-derived neutralizing antibody, AH100, by determining the HTNV glycoprotein Gn-AH100 antigen-binding fragment (Fab) complex structure. Our findings revealed that the epitopes situated on the domain A and b-ribbon and E3-like domain of the HTNV Gn head. By modeling the complex structure in the viral lattice, we propose that AH100 neutralizes the virus by impeding conformational changes of Gn protomer, which is crucial for viral entry. Additionally, sequence analysis of all reported natural isolates indicated the absence of mutations in epitope residues, suggesting the potential neutralization ability of AH100 in diverse isolates. Therefore, our results provide novel insights into the epitope and the molecular basis of AH100 neutralization.

Optimization of an adeno-associated viral vector for epidermal keratinocytes in vitro and in vivo

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

SUMO1 modification of 0N4R-tau is regulated by PIASx, SENP1, SENP2, and TRIM11

Tau is a microtubule-associated protein that contributes to cytoskeletal stabilization. Aggregation of tau proteins is associated with neurodegenerative disorders such as Alzheimer's disease. Several types of posttranslational modifications that alter the physical properties of tau proteins have been identified. SUMOylation is a reversible modification of lysine residues by a small ubiquitin-like modifier (SUMO). In this study, we examined the enzymes that regulate the SUMOylation and deSUMOylation of tau in an alternatively spliced form, 0N4R-tau. Among SUMO E3 ligases, we found protein inhibitor of activated STAT (PIAS)xα and PIASxβ increase the levels of SUMOylated tau. The deSUMOylation enzymes sentrin-specific protease (SENP)1 and SENP2 reduced the levels of SUMO-conjugated tau. SUMO1 modification increased the level of phosphorylated tau, which was suppressed in the presence of SENP1. Furthermore, we examined the effect of tripartite motif (TRIM)11, which was recently identified as an E3 ligase for SUMO2 modification of tau. We found that TRIM11 increased the modification of both 2N4R- and 0N4R-tau by SUMO1, which was attenuated by mutation of the target lysine residue to arginine. These findings suggest that the expression and activity of SUMOylation regulatory proteins modulate the physical properties of tau proteins and may contribute to the onset and/or progression of tau-associated neurodegenerative disorders.

Molecular basis of FIGNL1 in dissociating RAD51 from DNA and chromatin.

Maintaining genome integrity is an essential and challenging process. RAD51 recombinase, the central player of several crucial processes in repairing and protecting genome integrity, forms filaments on DNA. RAD51 filaments are tightly regulated. One of these regulators is FIGNL1, that prevents persistent RAD51 foci post-damage and genotoxic chromatin association in cells. The cryogenic electron microscopy structure of FIGNL1 in complex with RAD51 reveals that the FIGNL1 forms a non-planar hexamer and RAD51 N-terminus is enclosed in the FIGNL1 hexamer pore. Mutations in pore loop or catalytic residues of FIGNL1 render it defective in filament disassembly and are lethal in mouse embryonic stem cells. Our study reveals a unique mechanism for removing RAD51 from DNA and provides the molecular basis for FIGNL1 in maintaining genome stability.

Comparison of "framework Shuffling" and "CDR Grafting" in humanization of a PD-1 murine antibody.

Humanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans. Two different strategies of antibody humanization are popularly employed, including "complementarity determining region (CDR) grafting" and "framework (FR) shuffling" to humanize a murine antibody against human programmed death-1 (PD-1), XM PD1. In CDR-grafting humanization, the CDRs of XM PD-1, were grafted into the human FR regions with high homology to the murine FR counterparts, and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization, a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods, including antibody purity, thermal stability, binding efficacy, predicted humanness, and immunogenicity, along with T cell epitope prediction for the humanized antibodies. While the ideal molecule was not achieved through CDR grafting in this particular instance, FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background. The insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development, contributing to broader application and understanding in the field.

N-terminal acetylation of Set1-COMPASS fine-tunes H3K4 methylation patterns.

H3K4 methylation by Set1-COMPASS (complex of proteins associated with Set1) is a conserved histone modification. Although it is critical for gene regulation, the posttranslational modifications of this complex that affect its function are largely unexplored. This study showed that N-terminal acetylation of Set1-COMPASS proteins by N-terminal acetyltransferases (NATs) can modulate H3K4 methylation patterns. Specifically, deleting NatA substantially decreased global H3K4me3 levels and caused the H3K4me2 peak in the 5' transcribed regions to shift to the promoters. NatA was required for N-terminal acetylation of three subunits of Set1-COMPASS: Shg1, Spp1, and Swd2. Moreover, deleting Shg1 or blocking its N-terminal acetylation via proline mutation of the target residue drastically reduced H3K4 methylation. Thus, NatA-mediated N-terminal acetylation of Shg1 shapes H3K4 methylation patterns. NatB also regulates H3K4 methylation, likely via N-terminal acetylation of the Set1-COMPASS protein Swd1. Thus, N-terminal acetylation of Set1-COMPASS proteins can directly fine-tune the functions of this complex, thereby substantially shaping H3K4 methylation patterns.

The hydrophobicity of the CARD8 N-terminus tunes inflammasome activation

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

Toxin Homology Domain in Plant Type 2 Prolyl 4-Hydroxylases Acts as a Golgi Localization Domain.

Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain.

An activity-specificity trade-off encoded in human transcription factors

Transcription factors (TFs) control specificity and activity of gene transcription, but whether a relationship between these two features exists is unclear. Here we provide evidence for an evolutionary trade-off between the activity and specificity in human TFs encoded as submaximal dispersion of aromatic residues in their intrinsically disordered protein regions. We identified approximately 500 human TFs that encode short periodic blocks of aromatic residues in their intrinsically disordered regions, resembling imperfect prion-like sequences. Mutation of periodic aromatic residues reduced transcriptional activity, whereas increasing the aromatic dispersion of multiple human TFs enhanced transcriptional activity and reprogramming efficiency, promoted liquid–liquid phase separation in vitro and more promiscuous DNA binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. We propose that rational engineering of amino acid features that alter phase separation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.

The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation

Mitochondrial dysfunction is a prominent hallmark of Alzheimer's disease (AD). The transcriptional coactivator PPARγ coactivator 1 (PGC-1a) has been identified as a key regulator of mitochondrial biogenesis and function. However, the precise structure/function relationship between PGC-1a and mitochondrial quality control remains incompletely understood. In this study, we investigated the impact of PGC-1a on AD pathology and its underlying mechanisms with a specific focus on mitochondrial axonal transport. Additionally, we generated two PGC-1α mutants by substituting leucine residues at positions 148 and 149 within the LKKLL motif or at positions 209 and 210 within the LLKYL motif with alanine. Subsequently, we examined the effects of these mutants on mutAPP-induced abnormalities in anterograde and retrograde axonal transport, disrupted mitochondrial distribution, and impaired mitophagy.Mutagenesis studies revealed that the LLKYL motif at amino acid position 209–210 within PGC-1α plays an essential role in its interaction with estrogen-related receptors (ERRα), which is necessary for restoring normal mitochondrial anterograde axonal transport, maintaining proper mitochondrial distribution, and ultimately preventing neuronal apoptosis. Furthermore, it was found that the Leu-rich motif at amino acids 209–210 within PGC-1α is crucial for rescuing mutAPP-induced impairment in mitophagy and loss of membrane potential by restoring normal mitochondrial retrograde axonal transport. Conversely, mutation of residues 148 and 149 in the LKKLL motif does not compromise the effectiveness of PGC-1α. These findings provide valuable insights into the molecular determinants governing specificity of action for PGC-1α involved in regulating mutAPP-induced deficits in mitochondrial axonal trafficking. Moreover, they suggest a potential therapeutic target for addressing Alzheimer's disease.

Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

P-268 Unique Argonaute isoforms from mammalian oocytes: unveiling their true purpose

Abstract Study question How does the mouse oocyte-specific AGO2 isoform (AGO2ooc) affect RNA interference (RNAi) and microRNA (miRNA) pathways? Summary answer Mouse AGO2ooc presents reduced RNAi-like endonucleolytic cleavage of perfectly complementary targets. Functional analysis of its impact on fertility and oocyte transcriptome control is in progress. What is known already Argonaute proteins play a pivotal role in effector complexes dedicated to miRNA and RNAi pathways, where small RNAs mediate sequence-specific repression of target RNAs. While all mammalian AGO proteins (AGO1 to AGO4) can function in the miRNA pathway, inhibiting target translation, AGO2 uniquely engages in the RNAi pathway, directing the endonucleolytic cleavage of targets perfectly complementary to small RNAs. In mammalian somatic cells, miRNAs are part of a crucial posttranscriptional regulation network, while RNAi is mostly non-functional. Mouse oocytes, however, deviate from this norm: miRNA regulation proves dispensable, while the abrogation of RNAi results in meiotic spindle defects and sterility. Study design, size, duration Experimental study. Participants/materials, setting, methods We pooled 25 fully-grown GV oocytes in triplicates from non-stimulated 10-12 weeks-old C57BI/6NCrl females for RNA isolation and sequencing. To assess RNAi-like and miRNA-like activity, we transfected Ago2ooc/ooc clones and WT cells with NanoLuc luciferase reporters bearing one perfect complementary (1xP, RNAi-like), four bulged (4xB, miRNA-like) or four mutated (4xM, control) sites for Let-7a and miR-30c. A firefly luciferase reporter without any site was co-transfected for normalization purposes. Main results and the role of chance RNA sequencing revealed an alternative exon-1 in mouse oocyte AGO2, producing an isoform (AGO2ooc) with different amino acid composition in the N-terminus. Interestingly, the somatic version of this N-terminus presents amino acid residues known to be post-translationally modified. These residues are absent in AGO2ooc. To functionally analyze the oocyte-specific N-terminus, we genetically engineered NIH3T3 fibroblasts to express the oocyte alternative exon-1 (Ago2ooc/ooc). Notably, AGO2ooc-expressing cells presented reduced RNAi-like (2-5 fold) but indistinguishable miRNA-like activity when compared to WT cells in reporter assays for Let-7a and miR-30c. The lower RNAi-like activity presented by AGO2ooc-expressing cells was further confirmed in an independent RNAi assay employing the expression of dsRNA hairpins. Both isoforms showed similar abundance in fibroblasts and similar cleavage activity in vitro. Next, we wondered whether AGO2ooc also displays reduced RNAi activity in vivo. To answer that, we generated mouse models whose oocytes would express the somatic AGO2 isoform instead of the AGO2ooc, or N-terminal variants with mutations in putative key amino acid residues. These models will be further utilized to assess fertility and oocyte transcriptome. Limitations, reasons for caution We must investigate whether AGO2ooc also displays differential RNAi activity in mouse oocytes and is relevant for oocyte competence, using the developed mouse models. Wider implications of the findings Mouse oocytes are known to exceptionally operate RNAi. Remarkably, AGO2, the effector nuclease associated with RNAi-like target cleavage has a different N-terminus in oocytes, suggesting that this factor could have adapted to this atypical regulation. We hypothesize that AGO2ooc controls RNAi repression, gene expression and oocyte competence. Trial registration number not applicable

Mutations of Single Residues in the Complexin N-terminus Exhibit Distinct Phenotypes in Synaptic Vesicle Fusion.

The release of neurotransmitters (NTs) at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Among those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind soluble N-ethylmaleimide sensitive factor attachment protein receptor complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin (Cpx) II. The N-terminus (amino acid 1-27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On the one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine-tunes the degree of spontaneous and evoked NT release.

Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients.

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T50 30) was increased from 55.12°C to 62.44°C, and Tm value was increased from 48.36°C to 53.18°C. Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

Activity and function of the endothelial sodium channel is regulated by the effector domain of MARCKS like protein 1 in mouse aortic endothelial cells.

The endothelial sodium channel (EnNaC) plays an important role in regulating vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoEC) by the actin cytoskeleton and lipid raft association protein myristoylated alanine-rich C-kinase substrate like protein 1 (MLP1). We hypothesized that mutation of specific amino acid residues within the effector domain of MLP1 or loss of association between MLP1 and the anionic phospholipid phosphate PIP2 would significantly alter membrane association and EnNaC activity in mAoEC. mAoEC transiently transfected with a mutant MLP1 construct (three serine residues in the effector domain replaced with aspartate residues) showed a significant decrease in EnNaC activity compared to cells transfected with wildtype MLP1. Compared to vehicle treatment, mAoEC treated with the PIP2 synthesis blocker wortmannin showed less colocalization of EnNaC and MLP1. In other experiments, Western blot and densitometric analysis showed a significant decrease in MLP1 and caveloin-1 protein expression in mAoEC treated with wortmannin compared to vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoEC. Taken together, our results suggest constitutive phosphorylation of MLP1 attenuates the function of EnNaC in aortic endothelial cells by a mechanism involving a decrease in association with MLP1 and EnNaC at the membrane, while deletion of PIP2 decreases MARCKS expression and overall membrane fluidity.

SWEET family transporters act as water conducting carrier proteins in plants.

Dedicated water channels are involved in the facilitated diffusion of water molecules across the cell membrane in plants. Transporter proteins are also known to transport water molecules along with substrates, however the molecular mechanism of water permeation is not well understood in plant transporters. Here, we show plant sugar transporters from the SWEET (Sugar Will Eventually be Exported Transporter) family act as water-conducting carrier proteins via a variety of passive and active mechanisms that allow diffusion of water molecules from one side of the membrane to the other. This study provides a molecular perspective on how plant membrane transporters act as water carrier proteins, a topic that has not been extensively explored in literature. Water permeation in membrane transporters could occur via four distinct mechanisms which form our hypothesis for water transport in SWEETs. These hypothesis are tested using molecular dynamics simulations of the outward-facing, occluded, and inward-facing state of AtSWEET1 to identify the water permeation pathways and the flux associated with them. The hydrophobic gates at the center of the transport tunnel act as a barrier that restricts water permeation. We have performed in silico single and double mutations of the hydrophobic gate residues to examine the changes in the water conductivity. Surprisingly, the double mutant allows the water permeation to the intracellular half of the membrane and forms a continuous water channel. These computational results are validated by experimentally examining the transport of hydrogen peroxide molecules by the AtSWEET family of transporters. We have also shown that the transport of hydrogen peroxide follows the similar mechanism as water transport in AtSWEET1. Finally, we conclude that similar water-conduction states are also present in other SWEET transporters due to the high sequence and structure conservation exhibited by this transporter family.