Biology subjects

Zheng, Z.

Publications and source records attributed to Zheng, Z..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Gene Editing and Aging: A Bibliometric Analysis of Global Trends and Frontier Themes (2015-2024)

ObjectiveThe accumulation of DNA damage and mutations is a key contributor to aging. Recent studies have shown that disrupting the Beclin 1-BCL2 autophagy regulatory complex through gene editing can extend lifespan in mice. The precise application of gene editing technologies offers a promising strategy for aging. This study conducted a bibliometric analysis to map the knowledge landscape of gene editing and aging. MethodsWe retrieved publications related to genome editing and aging from the Web of Science Core Collection, covering the period from 2015 to 2024. The data were analyzed using VOSviewer and R package Bibliometrix. These tools enabled us to identify the most productive researchers, journals, institutions, countries and visualized current trends, emerging research hotspots. ResultsA total of 982 publications on genome editing and aging were identified. The United States (n=285) and China (n=214) form a dual-core structure leading global output. Harvard University (n=116) emerged as the most prolific institution. Scientific Reports was the top-publishing journal, with 23 articles in 2024. ZHANG Y (n=12, citations=102, H-index=6) was identified as the most productive author. KIM Es 2017 publication in Nature Communications (TC=494, TC/year=54.9, NTC=9.33) has had a significant and ongoing impact. The analysis indicates that future directions will include CRISPR optimization and AI-assisted genomic analysis. ConclusionThis study presents the first comprehensive bibliometric analysis and visualization of the knowledge structure in gene editing and aging research up to 2024. It offers researchers a detailed overview of current developments, trends, and emerging frontiers in this rapidly evolving domain.

scientific communication and education↗

bioRxiv2025

The structure of phycobilisome with a bicylindrical core from the cyanobacterium Synechococcus elongatus PCC 7942

Phycobilisomes (PBSs) are the major light-harvesting complexes in the cyanobacteria and red algae and they consist of a central core and peripheral rods that are attached to the core. The PBS cores contain 2-5 allophycocyanin cylinders that are organized by ApcE. At the present, structures of PBS with tricylindrical and pentacylindrical cores have been determined while the structure of the PBS with a bicylindrical core is yet to be revealed. Here we report the cryo-EM structure of PBS with bicylindrical core from Synechococcus elongatus PCC 7942 (Synechococcus 7942) at 2.98-[A] resolution. Similar to the PBS with a tricylindrical core, six peripheral rods are attached to the core by the rod-core linker protein CpcG in the PBS of Synechococcus 7942 even though the core lacks the top AP cylinder, which is important for the attachment of peripheral rods to the tricylindrical cores. We found that the C-terminus of ApcE in the Synechococcus 7942 was involved in interacting with both CpcG and CpcB of a top peripheral rod, compensating for the absence of the top AP cylinder of the core and maintaining PBS stability. Analysis of the bilin distribution reveals that distance of excitation energy transfer from top peripheral rods to the terminal emitters is approximately 15% shorter compared to the PBS with tricylindrical cores. Although there are 30% fewer bilin chromophores in the Synechococcus 7942 PBS core compared with the tricylindrical core, the aromatic residue ring in the Synechococcus 7942 PBS core is conserved, supporting the suggestion that these aromatic residues from AP and linker proteins are critical to the energy transfer of PBS.

plant biology↗

bioRxiv2025

Ancient DNA Reveals Hominoid Evolution: Intermediate DNA Sequences and Advances in Molecular Paleontology

AbstractFossils and ancient crude pottery vessels function as physical "DNA containers," preserving oriDNA (original, in situ DNA) and accumulating eDNA (environmental DNA) over time. Ancient DNA (aDNA) serves as molecular fossils, chronicling evolutionaryhistory. Using "nano-affinitybead technology", we extracted and sequenced DNAfrom Lycoptera fossils in the Jehol region and a "round-bellied jar (Jar)" from the Erlitou period in Guangwu Town, yielding 236,545 primate sequences from the fossils and 86,908 from the pottery. We observed that the AFF value of DNA sequences negatively corre lates with species divergence time1, offering a quantitative measure of DNA preservation and host divergence. Some fragments distinct from modern genomic sequences, termed "intermediate DNAsequences" (IDS), have been identified. Many IDS exhibit an upper age limit, preserving characteristics of the last common ancestor (LCA) of the Hominidae and offering molecular insights into "Darwins puzzle". Among IDS, the SRRA subtype (Sequence Reversal and Rearrangement), identified in mRNA-coding exonic sequences, arises from the incorporation of a complementary antisense strand upstream of the sense strand, either adjacent to it or separated bya sequence interval. This introduces a novel post-transcriptional regulatory mechanism at the mRNA level, driven by SRRA, which presets hairpin structures and Indels in the UTR or CDS of exonic sequences, modulating gene variation. We propose: "SRRAs played a critical trial-and-error role in early Hominidae evolution, facilitating adaptive genomic changes, with some SRRA sequences later excised from exons", positioning SRRAas an evolutionary "genetic switch". Additionally, seven species -specific fragments (SSFs) of non-human primates (NHPs) linked to Asian Homo erectus were identified in the fossils, and pottery DNA reveale d sequences from tropical species (e.g., zebrass, oil palms), providing evidence of climate-driven local extinction and supporting paleo-ecological and paleo-environmental reconstruction. This method of analyzing aDNA from non-skeletal materials opens new avenues in paleontology, archaeology, and geology, guiding the tracing of ancient migration patterns and fossil searches. DNAfragments preserved within "DNA containers " exhibit an "old-few, new-many" turnover pattern, with many aDNA fragments displaying non-deamination. This evidence challenges prevailing perspectives, the authenticity criteria for aDNA, and the capabilities and scope of the traditional research method, necessitating a thorough reevaluation of the relevant knowledge framework. Furthermore, this study opens new opportunities for frontier research.

paleontology↗

bioRxiv2025

Alpha-synuclein aggregation and dopaminergic neuron death in a new mouse model of Parkinson disease expressing human full-length and C-terminally truncated 1-120 alpha-synuclein.

Parkinsons disease (PD) is neuropathologically characterized by the presence of Lewy bodies and Lewy neurites made of aggregated alpha-synuclein (aSyn). Several studies have shown that Lewy bodies contain C-terminally truncated aSyn which is more prone to fibrillation and enhances the aggregation of human full-length aSyn in vitro. Nevertheless, in vivo studies addressing whether and how human C-terminally truncated aSyn may exacerbate the pathological aggregation and toxicity of human full-length aSyn are currently lacking. In the present study we aimed to determine whether the co-expression of human full-length and 1-120 truncated aSyn would enhance human full-length aSyn aggregation and toxicity. To this end we generated and characterized two novel alpha- synuclein mouse models. Specifically, we first produced a mouse line expressing full-length human aSyn under its own promoter (BACo mice) in the absence of endogenous mouse aSyn. Then, the BACo mice were crossed with the previously described MI2 mice, which express human 1-120 truncated aSyn under the control of the tyrosine hydroxylase (TH) promoter on a mouse aSyn null backgroundin order to obtain the MI2BACo mice, which express both human 1-120 truncated and full-length aSyn in a mouse aSyn null background. We found that aSyn aggregation was present and increased with age in both mouse lines but was significantly higher in the MI2BACo mice. These mice exhibited progressive nigrostriatal accumulation of aSyn aggregates which were often composed of full-length and 1-120haSyn, the latter being specifically recognized by using a novel antibody raised against the 1-120haSyn. In addition, we observed that the presence of both truncated and full-length aSyn in MI2BACo mice led to a faster and more pronounced aSyn aggregation which associated with more severe dopaminergic striatal fiber degeneration and nigral neuron loss. Collectively, our results indicate that the expression of human C-terminally truncated aSyn exacerbates human full-length aSyn pathology and support that the novel MI2BACo transgenic mouse line represents an innovative experimental model to study the biological basis of PD and test novel therapeutic approaches.

neuroscience↗

bioRxiv2025

Object categorization based on abstract concepts by monkeys, humans, and vision models

Humans categorize objects at multiple levels of abstraction--animate versus inanimate, big versus small, and many other attributes. Despite its apparent challenge, the advent of deep neural networks (DNNs) has demonstrated that complex visual processing alone can support such classification without language or human-specific knowledge. This raises a natural question: to what extent can non-human primates, without language, perform such categorization? Although basic object-recognition behavior in monkeys such as similarity judgment has been extensively studied, their ability to classify objects across diverse rules remains poorly characterized. Here, we developed a task paradigm that enabled us to train monkeys on a large battery of binary classification tasks using natural object images, spanning more than 10 rules, such as animate versus inanimate, natural versus man-made objects, and mammalian versus non-mammalian animals. Monkeys acquired each rule in a few days, generalized the learned rules to new images, and exhibited error patterns consistent with human judgments. At the same time, their classification performance correlated more strongly with that of visual DNNs trained without language input, whereas human performance was better explained by language-informed DNNs. These results provide an important benchmark for the capacity of biological neural networks to perform image classification without language and human-specific knowledge.

neuroscience↗

bioRxiv2025

Multi-modal Monte Carlo MRI simulator of tissue microstructure

Monte Carlo simulation of MRI signals is a powerful tool for modelling and quantifying tissue microstructure. While these methods have been used in MRI for decades, they typically focus on one aspect of microstructure and one type of MRI contrast at a time. In this paper, we present a new Monte Carlo MRI (MCMR) simulator for investigating the impact of tissue microstructure on arbitrary MRI sequences. A key feature of the proposed simulator is that substrates can incorporate multiple microstructural features (diffusion, T1/T2 relaxation, membrane permeability, local off-resonance fields, surface relaxation and magnetisation transfer) simultaneously. This provides a single forward model from tissue microstructure to MRI signals that captures a broad range of contrasts typically considered to be of different MRI "modalities". We validate the results of the simulator by reproducing previous findings in well-established sequences, namely diffusion-weighted MRI, magnetisation transfer imaging, and gradient and spin echo sequences. The simulator is fully open source and packaged along with detailed documentation and tutorials.

neuroscience↗

bioRxiv2025

SIRT6 activator fucoidan extends healthspan and lifespan in aged wild-type mice

SIRT6 is a protein deacylase, deacetylase, and mono-ADP-ribosylase (mADPr) regulating biological pathways important for longevity including DNA repair and silencing of LINE1 retrotransposons. SIRT6 knockout mice die by 30 days of age, whereas SIRT6 overexpression increases lifespan in male mice. Finding safe pharmacological activators of SIRT6 would have clinical benefits. Fucoidan, a polysaccharide purified from brown seaweed, has been identified as an activator of SIRT6 deacetylation activity. Here, we show that fucoidan also activates SIRT6 mADPr activity, which was shown to be elevated in certain human centenarians. Administering fucoidan to aged mice led to a significant increase in median lifespan in male mice. Both male and female mice demonstrated a marked reduction in frailty and epigenetic age. Fucoidan-treated mice showed repression of LINE1 elements suggesting that the beneficial effects of fucoidan are mediated, at least in part, by SIRT6. As brown seaweed rich in fucoidan is a popular food item in South Korea and Japan, countries with the highest life expectancy, we propose that fucoidan supplementation should be explored as a safe strategy for activating SIRT6 and improving human healthspan and lifespan.

molecular biology↗

bioRxiv2025

AI-Driven Efficient De Novo design of GLP-1RAs with Extended Half-Life and Enhanced Efficacy

Peptide drug has revolutionized modern therapeutics, offering novel treatment avenues for various diseases. However, low efficacy, time consumption, and high cost hinder peptide drug design and discovery. We present an efficientin approach that integrates deep learning-based protein design with efficient functional screening, enabling the rapid design of biotechnologically important peptides with improved the stability and efficacy. We designed 10,000 de novo GLP-1 receptor agonists (GLP-1RAs), of which 60 met the stability, efficacy, and diversity criteria in the vitral functional screening. In vitro validation revealed a 62% success rate, while in vivo experiment demonstrated that two designed GLP-1RAs exhibited significantly extended half-lives, approximately three times longer than Semaglutide. In diabetic and obesity mouse models, the most competitive candidate showed superior therapeutic efficacy over Semaglutide. Our AI driven peptide design pipline integrats protein design, functional screening, and experiment validation, reducing the number of iterations required to find novel peptide candidates. The entire process, from design to screening, can be completed in a single cycle within two weeks.

bioengineering↗

bioRxiv2025

Restoration of tumor suppressor protein with enhanced activity using engineered tRNAs to induce tumor regression

Cancer develops through a gradual accumulation of mutations including nonsense mutations in tumor suppressor genes (TSGs), which drive tumor proliferation and progression due to the impairment or complete loss of tumor suppressor protein function. The ability to restore functional tumor suppressor protein from mutant driver TSGs is a longstanding aspiration in cancer therapy, however, current strategies face formidable challenges in recovering proteins harboring heterogeneous mutations and circumventing dominant-negative effects exerted by endogenous mutant proteins. Therefore, the development of therapeutic interventions that are effective and generalizable across diverse nonsense mutations in cancer, to rescue endogenous mutant tumor suppressor proteins remains an urgent yet unmet need. Here we find that engineered suppressor transfer RNA (sup-tRNA) could overcome nonsense mutations in key driver tumor suppressor genes, such as TP53, thereby avoiding the dominant-negative effects through the restoration of endogenous mutant p53 with its original functions. Optimized sup-tRNA that we named super tRNA, enabling high-fidelity incorporation of desired amino acids, can further enhanced the transcriptional activity of p53 to suppress tumor cell proliferation. Moreover, we also establish a computational model to guide super tRNAs design, and demonstrate that super tRNA could effectively inhibit tumor growth by targeted readthrough of nonsense mutations in different tumor suppressor genes. In two xenograft models harboring nonsense mutant TP53, we observed up to a 70% reduction in tumor volume after lipid nanoparticle (LNP)-based sup-tRNA treatment, along with robust recovery of p53 expression and function, and no hematological or histological abnormalities detected. Our findings demonstrate the potential of sup-tRNA as a feasible and effective strategy to rescue and enhance tumor suppressor protein function, thereby preventing cancer development. As a novel therapeutic RNA, sup-tRNA provides a flexible strategy for targeting driver mutations in cancer, thereby expanding the potential of tRNA-based therapy in precision oncology.

bioengineering↗

bioRxiv2025

FveTRM5 plays a critical role in regulating fruit shape in woodland strawberry

Cultivated strawberry is a globally important fruit crop with high economic value. Fruit shape contributes to fruit quality and diversity and is a target for breeding, but very few regulatory genes have been reported in strawberry. Here, we identified an ethyl methanesulfonate (EMS) round fruit (rf) mutant that produces round or flat fruits in woodland strawberry. The primary candidate point mutation is located in the second exon of FvH4_2g22810, causing a premature stop codon at residue 266. This gene encodes a protein with a high similarity to TON1 RECRUITING MOTIF 5 (TRM5) and has therefore been named FveTRM5. Transformation of FveTRM5pro:FveTRM5 into rf could rescue the round fruit phenotype, suggesting that FveTRM5 is responsible for rf. Overexpression of FveTRM5 produced elongated organs in both Arabidopsis and woodland strawberry, suggesting a conserved role in different species. FveTRM5 is ubiquitously expressed with higher levels in developing organs. Observation of cell shape showed that FveTRM5 promotes cell elongation and inhibits cell division in the medial-lateral direction in the receptacle. The FveTRM5 protein localized to microtubules. In conclusion, our results suggest that FveTRM5 plays an essential role in regulating strawberry fruit shape by influencing cell elongation and cell division, providing an excellent target gene for breeding new fruit shape cultivars.

plant biology↗

bioRxiv2025

ClairS-TO: A deep-learning method for long-read tumor-only somatic small variant calling

Accurate identification of somatic variants in tumor is crucial but challenging, and typically requires a matched normal sample for reliable detection, which is often unavailable in real-world research and clinical scenarios, necessitating proficient algorithms to tell real somatic variants from germline variants and background noises. However, existing tumor-only somatic variant callers that were designed for short-read data dont work well with long-read. To fill the gap, we present ClairS-TO, a deep-learning-based method for long-read tumor-only somatic variant calling. ClairS-TO uses an ensemble of two disparate neural networks that were trained from the same samples but for opposite tasks - how likely/not likely a candidate is a somatic variant. ClairS-TO also applies multiple post-calling filters, including 1) nine hard-filters, 2) four public plus any number of user-supplied PoNs, and 3) a module that statistically separates somatic and germline variants using tumor purity and copy number profile. Benchmarks using COLO829 and HCC1395 show that ClairS-TO outperforms DeepSomatic in long-read. ClairS-TO is also applicable to short-read and outperforms Mutect2, Octopus, Pisces, and DeepSomatic. Extensive experiments across various sequencing coverages, VAF ranges, and tumor purities support that ClairS-TO has a broad coverage of usage scenarios. ClairS-TO is open-source, available at https://github.com/HKU-BAL/ClairS-TO.

bioinformatics↗

bioRxiv2025

Epithelial-mesenchymal transition couples with cell cycle arrest at various stages

Numerous computational approaches have been developed to infer cell state transition trajectories from snapshot single-cell data. Most approaches first require projecting high-dimensional data onto a low-dimensional representation; however, this can distort the dynamics of the system. Using epithelial-to-mesenchymal transition (EMT) as a test system, we show that both biology-guided low-dimensional representations and trajectory simulations in high-dimensional state space, not representations obtained with brute force dimensionality-reduction methods, reveal two broad paths of TGF-{beta} induced EMT. The paths arise from the coupling between cell cycle and EMT at either the G1 or G2/M phase, contributing to cell-cycle related EMT heterogeneity. Subsequent multi-plex immunostaining studies confirmed the multiple predicted paths at the protein level. The present study highlights the heterogeneity of EMT paths, emphasizes that caution should be taken when inferring transition dynamics from snapshot single-cell data in two- or three-dimensional representations, and shows that incorporating dynamical information can improve prediction accuracy.

systems biology↗

bioRxiv2025

LivecellX: A Deep-learning-based, Single-Cell Object-Oriented Framework for Quantitative Analysis in Live-Cell Imaging

Live-cell imaging uniquely captures single-cell dynamics in space and time, but robust analysis is limited by segmentation and tracking errors that accumulate across frames. We present LivecellX, a deep-learning-based pipeline that integrates instance-level segmentation error correction with trajectory refinement, leveraging temporal context to recover accurate cell tracks. LivecellX also introduces a benchmark dataset with detailed annotations of common error classes, providing a resource for method development and evaluation. Beyond error correction, the framework incorporates modules for classifying biological processes, reconstructing cell lineages, and analyzing dynamic behaviors. Users can interact with the system programmatically or through a Napari-based graphical interface, enabling flexible integration into diverse workflows. By coupling error-aware correction with comprehensive lineage and dynamics analysis, LivecellX establishes an open, extensible platform that advances the accuracy and scalability of live-cell imaging studies.

biophysics↗

bioRxiv2025

Lysogen formation governs colonies while lytic infection is more prevalent in single cells of the bloom-forming cyanobacterium, Microcystis

While the bloom-forming cyanobacterium Microcystis can exist as free-living single cells or within dense mucilaginous colonies, the drivers and consequences of colony formation remain unclear. Here, we integrated metatranscriptomic datasets from two Microcystis bloom events in Lake Taihu, China, to analyze and validate the functional differences between colonial and single-cell Microcystis. Our results confirmed colony expression profiles were disproportionately enriched in Microcystis transcripts (and functions) compared to other prokaryotic taxa. Concomitantly, viral infection strategies diverged by Microcystis community morphology: colony-associated cells expressed lysogeny-associated genes, while single cells exhibited increased signatures of lytic infection. These data are consistent with the hypothesis that Microcystis colonies foster conditions favorable to lysogen formation--likely due to local high cell densities and the resulting advantage of superinfection immunity--whereas solitary cells experience stronger lytic pressure. On a broader scale, our findings refine the understanding of bloom dynamics by identifying how community morphological states coincide with distinct host-virus interactions. Cumulatively, this work underscores the importance of colony formation in shaping Microcystis ecology and highlights the need for mechanistic studies that disentangle the interplay between phage infection modes, colony formation, and microbial community structure.

microbiology↗

bioRxiv2025

A novel virus vB_PshM_Y4 with low similarity to both cultured and uncultured viruses

Viruses are the abundant and diverse biological entities on the Earth. During past decades, our knowledge about genomic diversity of viruses has been greatly expanded through high-throughput sequencing. However, whether traditional isolation method could detect virus that could not be identified by high-throughput sequencing is still unknown. In this study, a novel virus, vB_PshM_Y4, infecting Pseudoalteromonas shioyasakiensis, which is an opportunistic pathogen of economically important marine species, was isolated. After comparing vB_PshM_Y4 with the over 15 million genomes of both cultivated and uncultivated viruses in the NCBI and IMG/VR v4 datasets, no similar genome was identified. This suggests that viral isolation methods can detect novel viruses that may not be identified through high-throughput sequencing. Transmission electron microscopy showed that vB_PshM_Y4 exhibits the myoviral morphology. The virus possesses a double-stranded DNA genome that is 158,231 bp in length, with a G+C content of 30.92%, and contains 226 open reading frames (ORFs). Six ORFs were identified as putative auxiliary metabolic genes (AMGs). Phylogenetic tree and gene content-based network analyses indicated that vB_PshM_Y4 may represent a novel viral species within an unknown family of the Caudoviricetes class. Additionally, biogeographical distribution analysis showed that vB_PshM_Y4 were mainly detected in the Arctic and temperate and tropical epipelagic zones with low abundance. ImportanceThis study suggests that viral isolation could detect novel virus that could not be identified through high-throughput sequencing, contributed to a deeper understanding of the genetic diversity and distribution of Pseudoalteromonas viruses, and offered valuable insights into the prevention and control of opportunistic pathogenic bacteria affecting economically important marine organisms.

microbiology↗

bioRxiv2025

Aberrant Modular Dynamics of Functional Networks in Schizophrenia and Their Relationship with Neurotransmitter and Gene Expression Profiles

IntroductionNumerous studies have emphasized the time-varying modular architecture of functional brain networks and its relevance to cognitive functions in healthy participants. However, how brain modular dynamics change in schizophrenia and how these alterations relate to neurotransmitter and transcriptomic signatures have not been well elucidated. MethodsWe harmonized resting-state fMRI data from a multi-site sample including 223 patients and 279 healthy controls and applied the multilayer network method to estimate the regional module switching rate (flexibility) of functional brain connectomes. We examined aberrant flexibility in patients relative to controls and explored its relations to neurotransmitter systems and postmortem gene expression. ResultsCompared with controls, patients with schizophrenia had significantly higher flexibility in the somatomotor and right visual regions, and lower flexibility in the left parahippocampal gyrus, right supramarginal gyrus, right frontal-operculum-insula, bilateral precuneus posterior cingulate cortex, and bilateral inferior parietal gyrus. These alterations were associated with multiple neurotransmitter systems and weighted gene transcriptomic profiles. The most relevant genes were preferentially enriched for biological processes of transmembrane transport and brain development, specific cell types, and previously identified schizophrenia-related genes. ConclusionsThis study reveals aberrant modular dynamics in schizophrenia and its relations to neurotransmitter systems and schizophrenia-related transcriptomic profiles, providing insights into the understanding of the pathophysiology underlying schizophrenia.

neuroscience↗

bioRxiv2025

Melatonin attenuates kidney injury by alleviating lysosomal damage in diabetic kidney disease

Proteinuria-induced damage to renal tubular epithelial cells is one of the main causes of diabetic kidney disease (DKD), and the clearance of overloaded albumin by lysosomes is crucial for maintaining the homeostasis of renal tubular epithelial cells. Therefore, lysosomal damage is closely related to the pathogenesis of DKD, but effective prevention and treatment measures are still lacking. Melatonin (MLT) is secreted by the pineal gland and can not only regulate circadian rhythms but also maintain lysosomal homeostasis. In this study, we demonstrate the presence of significant lysosomal damage in renal tubules of DKD patients, which causes autophagy impairment and a concomitant oxidative stress imbalance; however, MLT can upregulate transcription factor EB (TFEB) to improve lysosomal damage and restore the biosynthesis in this organelle. Mechanistically, MLT may protect lysosomes via the upregulation of TFEB and the miR-205-5p-LRP-1 pathway in renal tubules, thus improving autophagy dysfunction and oxidative imbalance in DKD.

pathology↗

bioRxiv2024

AtKC1 inhibits AtAKT1 activation via its amino-terminal inhibitory domain

AKT1 is a plant Shaker-like, hyperpolarization-activated K+ channel which plays a crucial role in K+ absorption. Besides phosphorylation, AKT1 is subject to negative regulation by AtKC1, a silent channel of the same family. Previous structural studies unveiled that AKT1 and AtKC1 form 2:2 heterotetramer in purified samples. However, the structural analysis failed to offer more insight into the inhibition mechanism of AKT1 activation by AtKC1. Here, inspecting the complex structure of AKT1-AtKC1 reveals that a stable domain of AtKC1 (residues 53 to 80), named inhibitory domain or I-domain, may inhibit AKT1 activation by stabilizing its depolarized, closed conformation. We confirmed this hypothesis with electrophysiological experiments. Interestingly, a single-point mutation (G315D) in AtKC1 has been reported to abolish its ability to inhibit AKT1. We solved the structure of AKT1-AtKC1(G315D) at a resolution of 2.8 [A] which revealed an unexpected stoichiometry alteration between AKT1 and AtKC1 from 2:2 to 3:1. This stoichiometry change further supports the hypothesis as we reason that single I-domain of AtKC1 in the channel complex is insufficient to effectively inhibit channel activation. Our findings reveal the inhibition mechanism of AtKC1 on AKT1 and offer insight into the regulatory mechanisms of hyperpolarization-activated channels.

biochemistry↗