Biology subjects

Zhao, Z.

Publications and source records attributed to Zhao, Z..

At least 325 records · Page 18Linked to original sources

bioRxiv2023

Genetic Inactivation of Beta-Catenin Attenuates and Its Activation Aggravates Desmoplakin Cardiomyopathy

AimMutations in the DSP gene encoding desmoplakin, a constituent of the desmosomes at the intercalated discs (IDs), cause a phenotype that spans arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy (DCM). It is typically characterized by biventricular enlargement and dysfunction, severe myocardial fibrosis, cell death, and arrhythmias. The canonical WNT (cWNT)/{beta}-catenin signaling pathway is implicated in the pathogenesis of ACM. Given that {beta}-catenin, an indispensable co-transcriptional regulator of the cWNT pathway, is also a member of the IDs, we genetically inactivated or activated {beta}-catenin to determine its role in the pathogenesis of the desmoplakin cardiomyopathy. Methods and ResultsThe Dsp gene was conditionally deleted in cardiac myocytes concomitant with the genetic inactivation or activation of {beta}-catenin using the tamoxifen-inducible MerCreMer mice. Inactivation and activation of {beta}-catenin were achieved upon deletion of its transcriptional domain and degrons, respectively. Analysis of cardiac myocytes transcripts and proteins showed marked dysregulation of the cWNT/{beta}-catenin pathway in the DSP-deficient mouse cardiac myocytes (Myh6-McmTam:DspF/F), as indicated by increased expression of cWNT/{beta}-catenin targets along with its inhibitors and isoforms of its key co-effectors. Genetic inactivation of {beta}-catenin in the Myh6-McmTam:DspF/F mice prolonged survival, improved cardiac function, reduced cardiac arrhythmias, and attenuated myocardial fibrosis, and cell death caused by apoptosis, necroptosis, pyroptosis, i.e., PANoptosis, whereas its activation had the opposite effects. Inactivation of {beta}-catenin was associated with partial restoration of the suppressed genes involved in OXPHOS, whereas its activation has the opposite effect. The beneficial effects were independent of the changes in the transcript levels of the cWNT target genes. ConclusionThe cWNT/{beta}-catenin was markedly dysregulated in the cardiac myocytes from a mouse model of DC. Inactivation of {beta}-catenin attenuated the phenotype partly through the recovery of OXPHOS genes whereas its activation had deleterious effects. The findings suggest suppression of {beta}-catenin might be beneficial in desmoplakin-cardiomyopathy. SummaryGenetic inactivation of {beta}-catenin improved desmoplakin cardiomyopathy, in part through the restoration of expression of genes involved in oxidative phosphorylation, whereas its activation was deleterious.

genetics↗

bioRxiv2023

A molecularly distinct accumbal-to-lateral hypothalamic circuit modulates food seeking and consumption

Orchestrating complex behavioral states, such as approach and consumption of food, is critical for survival. In addition to hypothalamus neuronal circuits, the nucleus accumbens (NAc) also plays an important role in controlling appetite and satiety in responses to changing external stimuli. However, the specific neuronal subtypes of NAc involved as well as how the humoral and neuronal signals coordinate to regulate feeding remain incompletely understood. Here, we deciphered the spatial diversity of neuron subtypes of the NAc shell (NAcSh) and defined a dopamine receptor D1(Drd1)- and Serpinb2-expressing subtype located in NAcSh encoding food consumption. Chemogenetics- and optogenetics-mediated regulation of Serpinb2+ neurons bidirectionally regulates food seeking and consumption specifically. Circuitry stimulation revealed the NAcShSerpinb2[->]LHLepR projection controls refeeding and can overcome leptin-mediated feeding suppression. Furthermore, NAcSh Serpinb2+ neuron ablation reduces food intake and upregulates energy expenditure resulting in body weight loss. Together, our study reveals a neural circuit consisted of molecularly distinct neuronal subtype that bidirectionally regulates energy homeostasis, which can serve as a potential therapeutic target for eating disorders.

neuroscience↗

bioRxiv2023

Systematic Analysis of KRAS-ligand Interaction Modes and Flexibilities Reveals the Binding Characteristics

KRAS, a common human oncogene, has been recognized as a critical drug target in treating multiple cancers. After four decades of effort, one allosteric KRAS drug (Sotorasib) has been approved, inspiring more KRAS-targeted drug research. Here we provide the features of KRAS binding pockets and ligand-binding characteristics of KRAS complexes using a structural systems pharmacology approach. Three distinct binding sites (conserved nucleotide-binding site, shallow Switch-I/II pocket, and allosteric Switch-II/3 pocket) are characterized. Ligand-binding features are determined based on encoded KRAS-inhibitor interaction fingerprints. Finally, the flexibility of the three distinct binding sites to accommodate different potential ligands, based on MD simulation, is discussed. Collectively, these findings are intended to facilitate rational KRAS drug design.

bioinformatics↗

bioRxiv2023

Imaging Microglia Surveillance during Sleep-wake Cycles in Freely Behaving Mice

Microglia surveillance manifests itself as dynamic changes in cell morphology and functional remodeling. Whether and how microglia surveillance is coupled to brain state switches during natural sleep-wake cycles remain unclear. To address this question, we used miniature two-photon microscopy (mTPM) to acquire time-lapse high-resolution microglia images of the somatosensory cortex, along with EEG/EMG recordings and behavioral video, in freely-behaving mice. We uncovered fast and robust brain state-dependent changes in microglia surveillance, occurring in parallel with sleep dynamics and early-onset phagocytic microglial contraction during sleep deprivation stress. We also detected local norepinephrine fluctuation occurring in a sleep state-dependent manner. We showed that the locus coeruleus-norepinephrine system, which is crucial to sleep homeostasis, is required for both sleep state-dependent and stress-induced microglial responses and {beta}2-adrenergic receptor signaling plays a significant role in this process. These results provide direct evidence that microglial surveillance is exquisitely tuned to signals and stressors that regulate sleep dynamics and homeostasis so as to adjust its varied roles to complement those of neurons in the brain. In vivo imaging with mTPM in freely behaving animals, as demonstrated here, opens a new avenue for future investigation of microglia dynamics and sleep biology in freely behaving animals.

neuroscience↗

bioRxiv2023

Lipopolysaccharide integrity primes bacterial sensitivity to a cell wall-degrading intermicrobial toxin

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the molecular basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, surface lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of lipopolysaccharide synthesis provided Escherichia coli (Eco) with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study highlights the consequences of co-regulating essential pathways on recipient fitness during interbacterial competition, and how antibacterial toxins leverage cellular vulnerabilities that are both direct and indirect to their specific targets in vivo.

microbiology↗

bioRxiv2023

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

CRISPR-Cas13 exclusively targets RNA. In prokaryotic cells, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. To investigate collateral cleavage by Cas13 in eukaryotic cells, we first compared various Cas13 orthologs and found that specifically LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage started within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulated genes associated with stress and innate immune response, ultimately leading to apoptotic cell death. This enabled us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, we used Nanopore sequencing to explore the identity of collaterally cleaved RNAs, the nucleotide position at which they are cleaved, and the temporal dynamics of collateral RNA cleavage. This revealed that LbuCas13a activation leads to rapid and global cleavage of cytoplasmic RNAs at specific nucleotide positions. In conclusion, we here report that LbuCas13a has high collateral activity in human cells and describe the temporal dynamics of the collateral RNA cleavage, the cellular responses ultimately leading to apoptosis, how this can be exploited as a cell elimination tool, and the collateral cleavage preferences of LbuCas13a.

molecular biology↗

bioRxiv2023

A converged ubiquitin-proteasome pathway for the degradation of TOC and TOM tail-anchored receptors

In plants, thousands of nucleus-encoded proteins translated in the cytosol are sorted to chloroplasts and mitochondria by binding to specific receptors of the TOC (translocon at the outer membranes of chloroplasts) and the TOM (translocon at the outer membranes of mitochondria) complexes for import into those organelles. The degradation pathways for these receptors are unclear. Here, we discovered a converged ubiquitin-proteasome pathway for the degradation of Arabidopsis thaliana TOC and TOM tail-anchored receptors. The receptors are ubiquitinated by E3 ligase(s) and pulled from the outer membranes by the AAA+ ATPase CDC48, after which a previously characterized cytosolic protein, TTOP, binds to the exposed transmembrane domains (TMDs) at the C termini of the receptors and CDC48, and delivers these complexes to the 26S proteasome.

plant biology↗

bioRxiv2023

Wetting by biomolecular condensates increases membrane lipid packing and dehydration

Membrane wetting by biomolecular condensates recently emerged as a key phenomenon in cell biology, playing an important role in a diverse range of processes across different organisms. However, an understanding of the molecular mechanisms behind condensate formation and interaction with lipid membranes is still missing. To study this, we exploited the properties of the dyes ACDAN and LAURDAN as nano-environmental sensors in combination with phasor analysis of hyperspectral and lifetime imaging microscopy. Using glycinin as a model condensate-forming protein and giant vesicles as model membranes, we obtained vital information on the process of condensate formation and membrane wetting. Our results reveal that glycinin condensates display differences in water dynamics when changing the salinity of the medium as a consequence of rearrangements in the secondary structure of the protein. Remarkably, analysis of membrane-condensates interaction with protein as well as polymer condensates indicated a correlation between increased wetting affinity and enhanced lipid packing. This is demonstrated by a decrease in the dipolar relaxation of water across all membrane-condensate systems, suggesting a general mechanism to tune membrane packing by condensate wetting.

biophysics↗

bioRxiv2022

MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that govern their size and functions remains unclear. We show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation and cell cycle re-entry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actin contractility, decreases the stiffness of the bulge and HG, and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

genetics↗

bioRxiv2022

Functionally dissecting claustrum along anterior-posterior axis in anxiety and higher sensitivity to cocaine

Adolescent cocaine exposure (ACE) induce anxiety and higher sensitivity to drug addiction during adulthood. Here, we show that the claustrum is crucial for control of these two distinct psychiatric disorders in ACE mice. In the process of anxiety test, the CaMKII-positive neurons in median portion of claustrum (MClaustrum) were obviously triggered, and chemogenetic suppressing these neurons efficiently reduced ACE-induced anxiety in adulthood. While, the CaMKII-positive neurons in anterior portion of claustrum (AClaustrum) were obviously activated in response to cocaine-induced conditioned place preference (CPP), and chemogenetic suppressing these neurons efficiently blocked cocaine CPP in ACE mice during adulthood. Our findings dissociating specific sub-portions of claustrum for drug-related anxiety and susceptibility of addiction, extending our understanding to diverse functions of claustrum subregions.

neuroscience↗

bioRxiv2022

Identification of S1PR4 as an immune modulator for favorable prognosis in HNSCC through unbiased machine learning

As the largest family of membrane proteins, G protein-coupled receptors (GPCRs) are the most prominent family of pharmacological targets. However, only a few GPCRs have been well-defined in terms of their physiological and pathological functions. Thus, an efficient way to identify key GPCRs involved in tumour formation is urgently needed. In this study, patients with head and neck squamous cell carcinoma (HNSCC) were classified into two different subtypes based on the characteristics of GPCRs using an unbiased machine learning method. Notably, these two subtypes showed significant differences in prognosis, gene expression, and immune microenvironment, especially in the infiltration of CD8+ T cells. Based on these differences, we screened three potential key regulators (S1PR4, S1PR5, and GPR87) from all GPCRs and constructed a prognostic nomogram for patients with HNSCC. We identified S1PR4 as the key GPCR in determining the two subtypes for positive correlation with the proportion and cytotoxicity of CD8+ T cells in HNSCC and was mainly expressed in a subset of CX3CR1+CD8+ T cells. We also demonstrated that S1PR4 is an immune modulator for the favourable prognosis of HNSCC patients. We found that S1PR4 was highly expressed in CD8+ T cells from the tumours of HNSCC patients, which was significantly associated with better prognosis, and S1PR4 expression was accompanied by higher T cell cytotoxic marker expression (IFNG and GZMB). Notably, S1PR4 co-localised with CX3CR1, which has been identified as the most cytotoxic marker of CD8+ T cells. Furthermore, S1PR4 upregulation could significantly increase T cell function in CAR-T cell therapy, indicating its great potential in cancer immunotherapy. Therefore, these results identified S1PR4 as a key indicator of cytotoxicity and the proportion of tumour-infiltrating CD8+ T cells and confirmed the prognostic value of S1PR4 in HNSCC. Our findings contribute to the knowledge of S1PR4 in anti-tumour immunity, providing a potential GPCR-targeted therapeutic option for future HNSCC treatment.

cancer biology↗

bioRxiv2022

Multi-modal multi-resolution atlas of the human neonatal cerebral cortex based on microstructural similarity

The neonatal period is a critical window for the development of the human brain and may hold implications for the long-term development of cognition and disorders. Multi-modal connectome studies have revealed many important findings underlying the adult brain but related studies were rare in the early human brain. One potential challenge is the lack of an appropriate and unbiased parcellation that combines structural and functional information in this population. Using 348 multi-modal MRI datasets from the developing human connectome project, we found that the information fused from the structural, diffusion, and functional MRI was relatively stable across MRI features and showed high reproducibility at the group level. Therefore, we generated automated multi-resolution parcellations (300 - 500 parcels) based on the similarity across multi-modal features using a gradient-based parcellation algorithm. In addition, to acquire a parcellation with high interpretability, we provided a manually delineated parcellation (210 parcels), which was approximately symmetric, and the adjacent areas around each boundary were statistically different in terms of the integrated similarity metric and at least one kind of original features. Overall, the present study provided multi-resolution and neonate-specific parcellations of the cerebral cortex based on multi-modal MRI properties, which may facilitate future studies of the human connectome in the early development period.

neuroscience↗

bioRxiv2022

Discovery and biological evaluation of a potent small molecule CRM1 inhibitor for its selective ablation of extranodal NK/T cell lymphoma

BackgroundThe overactivation of NF-{kappa}B signaling is a key hallmark for the pathogenesis of extranodal natural killer/T cell lymphoma (ENKTL), a very aggressive subtype of non-Hodgkins lymphoma yet with rather limited control strategies. Previously, we found that the dysregulated exportin-1 (also known as CRM1) is mainly responsible for tumor cells to evade apoptosis and promote tumor-associated pathways such as NF-{kappa}B signaling. MethodsHerein we reported the discovery and biological evaluation of a potent small molecule CRM1 inhibitor, LFS-1107. We validated that CRM1 is a major cellular target of LFS-1107 by biolayer interferometry assay (BLI) and the knockdown of CRM1 conferred tumor cells with resistance to LFS-1107. ResultsWe found that LFS-1107 can strongly suppresses the growth of ENKTL cells at low-range nanomolar concentration yet with minimal effects on human platelets and healthy peripheral blood mononuclear cells. Treatment of ENKTL cells with LFS-1107 resulted in the nuclear retention of IkB and consequent strong suppression of NF-{kappa}B transcriptional activities, NF-{kappa}B target genes downregulation and attenuated tumor cell growth and proliferation. Furthermore, LFS-1107 exhibited potent activities when administered to immunodeficient mice engrafted with human ENKTL cells. ConclusionsTherefore, LFS-1107 holds great promise for the treatment of ENKTL and may warrant translation for use in clinical trials.

pharmacology and toxicology↗

bioRxiv2022

Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from one to four days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.

microbiology↗

bioRxiv2022

Hydrodynamic and anthropogenic disturbances co-shape microbiota rhythmicity and community assembly within intertidal groundwater-surface water continuum

Tidal hydrodynamics drive the groundwater-seawater exchange and shifts in microbiota structure in the coastal zone. However, how the coastal water microbiota structure and assembly patterns respond to periodic tidal fluctuations and anthropogenic disturbance remain unexplored in the intertidal groundwater-surface water (GW-SW) continuum, although it affects biogeochemical cycles and coastal water quality therein. Here, through hourly time-series sampling in the saltmarsh tidal creek, rhythmic patterns of microbiota structure in response to daily and monthly tidal fluctuations in intertidal surface water are disentangled for the first time. The similarity in archaeal community structures between groundwater and ebb-tide surface water (R2=0.06, p=0.2) demonstrated archaeal transport through groundwater discharge, whereas multi-source transport mechanisms led to unique bacterial biota in ebb-tide water. Homogeneous selection (58.6%-69.3%) dominated microbiota assembly in the natural intertidal GW-SW continuum and the presence of 157 rhythmic ASVs identified at ebb tide and 141 at flood tide could be attributed to different environmental selection between groundwater and seawater. For intertidal groundwater in the tidal creek affected by anthropogenically contaminated riverine inputs, higher microbial diversity and shift in community structure were primarily controlled by increased co-contribution of dispersal limitation and drift (jointly 57.8%) and enhanced microbial interactions. Overall, this study fills the knowledge gaps in the tide-driven water microbial dynamics in coastal transition zone and the response of intertidal groundwater microbiota to anthropogenic pollution of overlying waters. It also highlights the potential of microbiome analysis in enhancing coastal water quality monitoring and identifying anthropogenic pollution sources (e.g., aquaculture pathogenic Vibrio) through the detection of rhythmic microbial variances associated with intertidal groundwater discharge and seawater intrusion.

ecology↗

bioRxiv2022

Single-cell multi-omics decodes regulatory programs during development of mouse secondary palate

The abnormal perturbation in gene regulation during palatogenesis may lead to cleft palate, a major congenital birth defect in humans and mice. However, a comprehensive multi-omic map of the developing secondary palate at single-cell resolution is lacking. In this study, we performed single-cell multiome sequencing and profiled chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. Application of optimal transport reconstructed five trajectories, representing continuous differentiation of multipotent cells into different subpopulations in later stages. By linking open chromatin signals to gene expression changes, we discovered a list of lineage-determining transcription factors, such as Shox2 for the anterior and Dlx1/2 for the posterior palatal mesenchymal trajectories. In conclusion, this study charted epigenetic and transcriptional dynamics during palatogenesis, which provides a valuable resource for the community and facilitate future research in cleft palate. HighlightsO_LIThe first report on building a single-cell multi-omics atlas with joint chromatin accessibility and gene expression measurements from the same cells during the development of mouse secondary palate. C_LIO_LIApplication of optimal transport calculated fate probabilities to different terminal states and recovered continuous landscapes during mouse secondary palate development. C_LIO_LIBy linking cis-regulatory DNA elements to target genes, we characterized a series of transcription factors governing the differentiation of cranial neural crest-derived multipotent cells to the anterior and posterior palatal mesenchymal trajectories, respectively. C_LIO_LITranscription factors Shox2 and Dlx1/2 exhibited top regulatory roles for the anterior and posterior palatal mesenchymal trajectories, respectively, showing significant enrichment in both motif accessibility and gene expression. C_LI

developmental biology↗

bioRxiv2022

Activation of innate immune cGAS-STING pathway contributes to Alzheimer pathogenesis in 5xFAD mice

cGAS senses microbial and host-derived double-stranded DNA (dsDNA) in cytoplasm to trigger cellular innate immune response in a STING-dependent manner. However, it remains unknown whether the cGAS-STING pathway in innate immunity contributes to Alzheimers disease (AD). Here we demonstrated the detectable binding of the cGAS-dsDNA in cytoplasm and the activation of microglial cGAS-STING pathway in brains of human AD and aged mice, suggesting a role for the cGAS-STING pathway in this neurodegenerative disease. Cgas-/-;5xFAD mice were largely protected from cognitive impairment, amyloid-{beta} (A{beta}) pathology, neuroinflammation and other sequelae associated with AD. Furthermore, Cgas deficiency in microglia inhibited the conversion of astrocytes into neurotoxic A1 phenotype, and thus alleviated oligomeric A{beta} peptides-induced neuronal toxicity. Finally, administration of STING inhibitor H-151 potently suppressed the activation of the cGAS-STING pathway and ameliorated AD pathogenesis in 5xFAD mouse model. In conclusion, our present study has identified a critical molecular link between innate immunity and AD, and suggests that therapeutic targeting of the cGAS-STING pathway activity might effectively interfere with the progression of AD.

neuroscience↗

bioRxiv2022

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

{beta}-lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of {beta}-lactamases among pathogenic bacteria limits the efficacy of {beta}-lactam antibiotics. This has created a major public health crisis. The use of {beta}-lactamase inhibitors has proven useful in restoring the activity of {beta}-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-{beta}-lactamases (MBLs) are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the {beta}-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, 3-{beta}7 and {beta}12-5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics (MD) simulations were performed, Markov State Models (MSM) were built, and convolutional variational autoencoder (CVAE)-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between 3-{beta}7 and {beta}12-5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.

biophysics↗