Comprehensive screening strategy coupled with structure-guided engineering of l-threonine aldolase from Pseudomonas putida for enhanced catalytic efficiency towards l-threo-4-methylsulfonylphenylserine.

l-Threonine aldolases (TAs) can catalyze aldol condensation reactions to form ß-hydroxy-α-amino acids, but afford unsatisfactory conversion and poor stereoselectivity at the Cß position. In this study, a directed evolution coupling high-throughput screening method was developed to screen more efficient l-TA mutants based on their aldol condensation activity. A mutant library with over 4000 l-TA mutants from Pseudomonas putida were obtained by random mutagenesis. About 10% of mutants retained activity toward 4-methylsulfonylbenzaldehyde, with five site mutations (A9L, Y13K, H133N, E147D, and Y312E) showing higher activity. Iterative combinatorial mutant A9V/Y13K/Y312R catalyzed l-threo-4-methylsulfonylphenylserine with a 72% conversion and 86% diastereoselectivity, representing 2.3-fold and 5.1-fold improvements relative to the wild-type. Molecular dynamics simulations illustrated that additional hydrogen bonds, water bridge force, hydrophobic interactions, and π-cation interactions were present in the A9V/Y13K/Y312R mutant compared with the wild-type to reshape the substrate-binding pocket, resulting in a higher conversion and Cß stereoselectivity. This study provides a useful strategy for engineering TAs to resolve the low Cß stereoselectivity problem and contributes to the industrial application of TAs.

NMDA Receptor-Arc Signaling Is Required for Memory Updating and Is Disrupted in Alzheimer's Disease.

BACKGROUND: Memory deficits are central to many neuropsychiatric diseases. During acquisition of new information, memories can become vulnerable to interference, yet mechanisms that underlie interference are unknown. METHODS: We describe a novel transduction pathway that links the NMDA receptor (NMDAR) to AKT signaling via the immediate early gene Arc and evaluate its role in memory. The signaling pathway is validated using biochemical tools and transgenic mice, and function is evaluated in assays of synaptic plasticity and behavior. The translational relevance is evaluated in human postmortem brain. RESULTS: Arc is dynamically phosphorylated by CaMKII (calcium/calmodulin-dependent protein kinase II) and binds the NMDAR subunits NR2A/NR2B and a previously unstudied PI3K (phosphoinositide 3-kinase) adapter p55PIK (PIK3R3) in vivo in response to novelty or tetanic stimulation in acute slices. NMDAR-Arc-p55PIK recruits p110α PI3K and mTORC2 (mechanistic target of rapamycin complex 2) to activate AKT. NMDAR-Arc-p55PIK-PI3K-mTORC2-AKT assembly occurs within minutes of exploratory behavior and localizes to sparse synapses throughout hippocampal and cortical regions. Studies using conditional (Nestin-Cre) p55PIK deletion mice indicate that NMDAR-Arc-p55PIK-PI3K-mTORC2-AKT functions to inhibit GSK3 and mediates input-specific metaplasticity that protects potentiated synapses from subsequent depotentiation. p55PIK conditional knockout mice perform normally in multiple behaviors including working memory and long-term memory tasks but exhibit deficits indicative of increased vulnerability to interference in both short-term and long-term paradigms. The NMDAR-AKT transduction complex is reduced in postmortem brain of individuals with early Alzheimer's disease. CONCLUSIONS: A novel function of Arc mediates synapse-specific NMDAR-AKT signaling and metaplasticity that contributes to memory updating and is disrupted in human cognitive disease.

Structure-based rational design of hydroxysteroid dehydrogenases for improving and diversifying steroid synthesis.

A group of steroidogenic enzymes, hydroxysteroid dehydrogenases are involved in steroid metabolism which is very important in the cell: signaling, growth, reproduction, and energy homeostasis. The enzymes show an inherent function in the interconversion of ketosteroids and hydroxysteroids in a position- and stereospecific manner on the steroid nucleus and side-chains. However, the biocatalysis of steroids reaction is a vital and demanding, yet challenging, task to produce the desired enantiopure products with non-natural substrates or non-natural cofactors, and/or in non-physiological conditions. This has driven the use of protein design strategies to improve their inherent biosynthetic efficiency or activate their silent catalytic ability. In this review, the innate features and catalytic characteristics of enzymes based on sequence-structure-function relationships of steroidogenic enzymes are reviewed. Combining structure information and catalytic mechanisms, progress in protein redesign to stimulate potential function, for example, substrate specificity, cofactor dependence, and catalytic stability are discussed.

Understanding gift-giving in game live streaming on Douyu: An evaluation of PSR/social presence.

In China, live streaming has grown rapidly in recent years, with gift-giving, a unique business model in live streaming, driving the development of many industries. This article explores the association between gift-giving behavior in game live streaming and viewers' live streaming experience. Specifically, this study aims to explore the correlation between Para-social Relationships, Social Presence, and gift-giving in the context of China. Based on the survey and interview of the viewer on Douyu, a Chinese live streaming platform, this study found that there is only a weak to a medium correlation between para-social relationships and viewers' gift-giving behavior. The correlation between social presence and gift-giving was even weaker. Although the conclusion of this study may be affected by the sample size limitation, it can still provide a reference for the current research on gift-giving on Chinese live streaming platforms.

MacRS controls morphological differentiation and natamycin biosynthesis in Streptomyces gilvosporeus F607.

Streptomyces gilvosporeus F607 produces large amounts of natamycin in a process regulated by multiple networks, including two-component systems (TCSs). The macR and macS genes, which are annotated as rs12540 and rs12545, respectively, in S. gilvosporeus F607, affect natamycin biosynthesis and sporulation. The findings of this study indicate that deletion of macRS from S. gilvosporeus F607 prevents the production of natamycin, delays spore formation (according to scanning electron microscopy), and results in aerial hyphae lacking compartments separated by septa (according to transmission electron microscopy). Real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that the expression levels of natamycin biosynthesis-related genes and genes essential for septum formation during sporulation were affected in the ΔmacRS mutant strain. Molecular simulations and electrophoretic mobility shift assays (EMSAs) suggested MacR not only interacted with the intergenic region of sgnM and sgnR, but also with the promoter of penicillin-binding protein gene ftsL required for cell division. sgnR promoter was presumed to be the binding target of MacR based on the RT-qPCR results. MacR had different affinity with two binding sites: one was located at ftsL promoter region with a perfect inverted repeats 'TGAGTACGCGTACTCA', the other was located at the presumed sgnR promoter with an imperfect inverted repeats 'TGAAGGTGCTGGACTCA'. We propose a hypothesis of a three-level regulatory pathway based on pleiotropic transcriptional regulator MacR and its target genes sgnR and ftsL; the pathway activates natamycin biosynthesis and influences septum development via direct and indirect effects in S. gilvosporeus F607.

Use of elicitors to enhance or activate the antibiotic production in streptomyces.

Streptomyces is the largest and most significant genus of Actinobacteria, comprising 961 species. These Gram-positive bacteria produce many versatile and important bioactive compounds; of these, antibiotics, specifically the enhancement or activation of their production, have received extensive research attention. Recently, various biotic and abiotic elicitors have been reported to modify the antibiotic metabolism of Streptomyces, which promotes the production of new antibiotics and bioactive metabolites for improvement in the yields of endogenous products. However, some elicitors that obviously contribute to secondary metabolite production have not yet received sufficient attention. In this study, we have reviewed the functions and mechanisms of chemicals, novel microbial metabolic elicitors, microbial interactions, enzymes, enzyme inhibitors, environmental factors, and novel combination methods regarding antibiotic production in Streptomyces. This review has aimed to identify potentially valuable elicitors for stimulating the production of latent antibiotics or enhancing the synthesis of subsistent antibiotics in Streptomyces. Future applications and challenges in the discovery of new antibiotics and enhancement of existing antibiotic production using elicitors are discussed.

Ile258Met mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase significantly enhances catalytic efficiency, cofactor affinity, and thermostability.

NAD(H)-dependent 7α-hydroxysteroid dehydrogenase catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. Here, we designed mutations of Ile258 adjacent to the catalytic pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase. The I258M variant gave a 4.7-fold higher kcat, but 4.5-fold lower KM, compared with the wild type, resulting in a 21.8-fold higher kcat/KM value for chenodeoxycholic acid oxidation. It presented a 2.0-fold lower KM value with NAD+, suggesting stronger binding to the cofactor. I258M produced 7-oxolithocholic acid in the highest yield of 92.3% in 2 h, whereas the wild-type gave 88.4% in 12 h. The I258M mutation increased the half-life from 20.8 to 31.1 h at 30 °C. Molecular dynamics simulations indicated increased interactions and a modified tunnel improved the catalytic efficiency, and enhanced rigidity at three regions around the ligand-binding pocket increased the enzyme thermostability. This is the first report about significantly improved catalytic efficiency, cofactor affinity, and enzyme thermostability through single site-mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase. KEY POINTS: • Sequence and structure analysis guided the site mutation design. • Thermostability, catalytic efficiency and 7-oxo-LCA production were determined. • MD simulation was performed to indicate the improvement by I258M mutation.

A new maltogenic amylase from Bacillus licheniformis R-53 significantly improves bread quality and extends shelf life.

Maltogenic amylase suppressed starch retrogradation in baked products. Here, a maltogenic amylase-producing strain of bacteria was screened and identified as Bacillus licheniformis R-53. Its coding gene was cloned and over-expressed in Bacillus subtilis WB600. Recombinant maltogenic amylase BLMA exhibited activity of 3235 U/mg under optimal conditions (60 °C and pH 6.5), with a good thermostability and pH stability. Mixolab experiment showed that a concentration of 60 ppm BLMA significantly improved the operating characteristics of dough. Baking test indicated the recombinant BLMA reduced bread hardness by 2.12 times compared with the control. Compared with maltogenic amylase from Novozymes (Novamyl 3D BG) and Angel Yeast Co. Ltd. (MAM100), BLMA has better effect on improving the bread volume, and almost the same effect on reducing hardness, improving elasticity and maintaining sensory as Novamyl 3D BG. Adding BLMA improved bread quality, increased bread volume and decreased hardness during storage, thus extending its shelf life.

The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

BACKGROUND: Carbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel blaOXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance. METHODS: A. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN. RESULTS: MICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene blaOXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer blaOXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants. CONCLUSIONS: Our investigation showed that A. johnsonii M19 is a source and disseminator of blaOXA-23 and carbapenem resistance. The ability to transfer blaOXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Arc Oligomerization Is Regulated by CaMKII Phosphorylation of the GAG Domain: An Essential Mechanism for Plasticity and Memory Formation.

Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.

Pd nanoparticle-decorated graphitic C3N4 nanosheets with bifunctional peroxidase mimicking and ON-OFF fluorescence enable naked-eye and fluorescent dual-readout sensing of glucose.

In this work, we report a novel naked-eye and fluorescent dual-readout glucose sensing platform based on the bifunctional peroxidase mimicking and ON-OFF fluorescence of Pd nanoparticle-decorated graphitic C3N4 nanosheets (PdNPs/g-C3N4). The PdNPs/g-C3N4 hybrid not only exhibited a strong peroxidase-like activity to catalyze colorless o-phenylenediamine (OPD) into its yellow product OPDox in the presence of H2O2, but it also acted as a turn-off fluorescent sensor for the assembly of the generated OPDox on g-C3N4. When coupled with glucose oxidase (GOx), the platform could provide both visual and fluorescent responses for glucose specifically. Under optimal conditions, the platform was able to detect the target at concentrations as low as 50 µM and 0.4 µM in the linear ranges of 50-2000 µM and 1-1000 µM with the naked eye and a fluorometer, respectively. The developed platform integrates the equipment-free merit of naked-eye observation with the excellent performance of fluorometric analysis well, providing a new and powerful tool for both rough assessment and accurate detection of glucose in diabetes management and other applications.

A molecularly-imprinted-electrochemical-sensor modified with nano-carbon-dots with high sensitivity and selectivity for rapid determination of glucose.

In this work, a novel molecularly imprinted electrochemical sensor (MIECS) based on a glassy carbon electrode (GCE) modified with carbon dots (CDs) and chitosan (CS) for the determination of glucose was proposed for the first time. The use of the environmental-friendly CDs and CS as electrode modifications improved the active area and electron-transport ability substantially, while 3-aminobenzeneboronic acid was used as a functional monomer and glucose as template for the fabrication of molecularly imprinted polymer (MIP) film to detect glucose via differential pulse voltammetry. Transmission electron microscope, Fourier transform infrared spectroscopy, energy dispersive x-ray spectrometry, cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were applied to characterize the fabricated sensor. Experimental conditions such as molar ratio of functional monomer to template, volume ratio of CDs to CS, incubation time and elution time were optimized. By using glucose as a model analyte, the MIECS had two assay ranges of 0.5-40 µM and 50-600 µM, and fairly low limit of detection (LOD) of 0.09 µM (S/N = 3) under the optimized conditions. The MIECS also exhibited excellent selectivity, good reproducibility, and stability. The proposed sensor was successfully applied to a preliminary test for glucose analysis in real human blood serum samples.

Uricase-free on-demand colorimetric biosensing of uric acid enabled by integrated CoP nanosheet arrays as a monolithic peroxidase mimic.

In clinical diagnosis, monitoring of uric acid (UA) is generally realized by combining uricase with natural peroxidase. The use of bio-enzymes, however, shadows some highlights of these methods due to their vulnerable activities against environments. Herein, we report a novel biosensor for the natural enzyme-free colorimetric detection of UA by using CoP nanosheet arrays grown on Ni foam (NF) as a monolithic peroxidase mimic. The integrated nanozyme can be put into and taken out from reaction systems conveniently with only tweezers, making it possible for on-demand analysis. As demonstrated, the obtained CoP/NF exhibits outstanding peroxidase-like activity to trigger the oxidation reaction of colorless 3,3'5,5'-tetramethylbenzidine (TMB) to a blue product (TMBox) mediated by H2O2. It is found that the blue TMBox can be reduced to colorless TMB again by UA selectively, thus the presence of UA in solutions will suppress the color reaction of TMB. Based on this principle, an uricase-free biosensor is developed for the photometric determination of UA, providing a wide detection range of 1-200 µM and a limit of detection down to 1.0 µM. In addition, the fabricated biosensor can be applied for measuring UA in clinical samples with merits of simple operation and good reliability, exhibiting its great promise in clinical diagnosis.

One-Pot Anchoring of Pd Nanoparticles on Nitrogen-Doped Carbon through Dopamine Self-Polymerization and Activity in the Electrocatalytic Methanol Oxidation Reaction.

Exploration of advanced electrocatalysts to promote the sluggish methanol oxidation reaction (MOR) is of vital importance for developing high efficiency and low-cost direct methanol fuel cells. Highly dispersed palladium nanoparticles (Pd NPs) anchored on a nitrogen-doped carbon support were fabricated using a facile one-pot dopamine self-polymerization mediated redox strategy, in which dopamine not only acted as a moderate reductant to induce the formation of Pd NPs during self-polymerization but was also the precursor of the nitrogen-doped carbon support for Pd. The synthesized hybrid features the following characteristics: 1) High dispersity of Pd NPs, which exposed a high abundance of active surfaces and sites for heterogeneous electrocatalysis; 2) metal-support interactions, which may affect the surface chemistry and electron distribution of active Pd NPs; 3) the Pd NPs were partially imbedded or encapsulated into the support, thus reducing the possible agglomeration of Pd NPs during cyclic measurements. The electrocatalyst with such favorable features provided higher mass activity (2.2 times that of commercial Pd/C) and better durability (reduced loss of activity during simulated frequent startup-shutdown operations) for the MOR in alkaline media.

Structural basis of arc binding to synaptic proteins: implications for cognitive disease.

Arc is a cellular immediate-early gene (IEG) that functions at excitatory synapses and is required for learning and memory. We report crystal structures of Arc subdomains that form a bi-lobar architecture remarkably similar to the capsid domain of human immunodeficiency virus (HIV) gag protein. Analysis indicates Arc originated from the Ty3/Gypsy retrotransposon family and was "domesticated" in higher vertebrates for synaptic functions. The Arc N-terminal lobe evolved a unique hydrophobic pocket that mediates intermolecular binding with synaptic proteins as resolved in complexes with TARPγ2 (Stargazin) and CaMKII peptides and is essential for Arc's synaptic function. A consensus sequence for Arc binding identifies several additional partners that include genes implicated in schizophrenia. Arc N-lobe binding is inhibited by small chemicals suggesting Arc's synaptic action may be druggable. These studies reveal the remarkable evolutionary origin of Arc and provide a structural basis for understanding Arc's contribution to neural plasticity and disease.

Crystal structure of human Gadd45γ [corrected] reveals an active dimer.

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45γ [corrected], revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45γ [corrected] identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45γ [corrected] is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.

Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity.

An (S)-specific carbonyl reductase (SCRII) was purified to homogeneity from Candida parapsilosis by following an anti-Prelog reducing activity of 2-hydroxyacetophenone. Peptide mass fingerprinting analysis shows SCRII belongs to short-chain dehydrogenase/reductase family. Its coding gene was cloned and overexpressed in Escherichia coli. The recombinant SCRII displays the similar enzymatic characterization and catalytic properties to SCR. It catalyzes the enantioselective reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with excellent optical purity of 100% in higher yield than SCR. Based on the sequence-structure alignment, several single-point mutations inside or adjacent to the substrate-binding loop or active site were designed. With respect to recombinant native SCRII, the A220 and E228 mutations almost lost enantioselectivity towards 2-hydroxyacetophenone reduction. The catalytic efficiencies (kcat/Km) for the A220 or E228 variants are <7% that of the unmutated enzyme. This work provides an excellent catalyst for enantiopure alcohol preparation and the lethal mutations of A220 and E228 suggest their importance in substrate-binding and/or catalysis.

Structure-functional analyses of CRHSP-24 plasticity and dynamics in oxidative stress response.

The cold shock domain (CSD) is an evolutionarily conserved nucleic acid binding domain that exhibits binding activity to RNA, ssDNA, and dsDNA. Mammalian CRHSP-24 contains CSD, but its structure-functional relationship has remained elusive. Here we report the crystal structure of human CRHSP-24 and characterization of the molecular trafficking of CRHSP-24 between stress granules and processing bodies in response to oxidative stress. The structure of CRHSP-24 determined by single-wavelength anomalous dispersion exhibits an α-helix and a compact ß-barrel formed by five curved anti-parallel ß strands. Ligand binding activity of the CSD is orchestrated by residues Ser(41) to Leu(43). Interestingly, a phosphomimetic S41D mutant abolishes the ssDNA binding in vitro and causes CRHSP-24 liberated from stress granules in vivo without apparent alternation of its localization to the processing bodies. This new class of phosphorylation-regulated interaction between the CSD and nucleic acids is unique in stress granule plasticity. Importantly, the association of CRHSP-24 with stress granules is blocked by PP4/PP2A inhibitor calyculin A as PP2A catalyzes the dephosphorylation of Ser(41) of CRHSP-24. Therefore, we speculate that CRHSP-24 participates in oxidative stress response via a dynamic and temporal association between stress granules and processing bodies.

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit.

Nascent polypeptide associated complex (NAC) and its two isolated subunits, αNAC and ßNAC, play important roles in nascent peptide targeting. We determined a 1.9 Å resolution crystal structure of the interaction core of NAC heterodimer and a 2.4 Å resolution crystal structure of αNAC NAC domain homodimer. These structures provide detailed information of NAC heterodimerization and αNAC homodimerization. We found that the NAC domains of αNAC and ßNAC share very similar folding despite of their relative low identity of amino acid sequences. Furthermore, different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer. In addition, we successfully built a ßNAC NAC domain homodimer model based on homologous modeling, suggesting that NAC domain dimerization is a general property of the NAC family. These 3D structures allow further studies on structure-function relationship of NAC.

Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione.

Eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, yet its biochemical functions remain elusive. We report the crystal structures of human LanCL1, both free of and complexed with glutathione, revealing glutathione binding to a zinc ion at the putative active site formed by conserved GxxG motifs. We also demonstrate by in vitro affinity analysis that LanCL1 binds specifically to the SH3 domain of a signaling protein, Eps8. Importantly, expression of LanCL1 mutants defective in Eps8 interaction inhibits nerve growth factor (NGF)-induced neurite outgrowth, providing evidence for the biological significance of this novel interaction in cellular signaling and differentiation.