Biology subjects

Zhai, Y.

Publications and source records attributed to Zhai, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling

Accurate chromosome segregation is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), to inhibit the anaphase-promoting complex or cyclosome (APC/C). Cdc20 is an essential mitotic regulator since it promotes mitotic exit through activating the APC/C and monitors kinetochore-microtubule attachment through activating the SAC. The proper functioning of Cdc20 requires multiple interactions with APC/C and MCC subunits. To functionally assess each of these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNAi. Here we generated Cdc20 RNAi sensitive cell lines by CRISPR/Cas9 which display a penetrant metaphase arrest phenotype by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron was found to be critical for the SAC by promoting the MCC formation and stabilizing the interaction between the MCC and APC/C. These data reveal additional regulatory components within the SAC and establish a novel method to interrogate Cdc20 function.

cell biology↗

bioRxiv2023

Pathogen protein modularity enables elaborate mimicry of a host phosphatase

Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruit the Serine/Threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.

plant biology↗

bioRxiv2023

Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to Serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus evolution. Author SummaryThe Spike strain of SARS-CoV-2 has accumulated many mutations during its time in circulation, most of which have occurred in the S1 region, and more specifically in the RBD, in an effort to either improve the viruss affinity for the receptor ACE2 or to enhance its ability to evade the immune system. Mutations in the Spike S2 region have more far-reaching effects than those in the S1 region because it is more conserved across sarbecoviruses. By comparing SARS and SARS2, we found that an important substitution at amino acid position 813 in the S2 region (T813S) disrupts the utilization of TMPRSS2 and can significantly influence viral entry into cells. This discovery deepens our knowledge of S proteins and provides new prospects for tracing the evolution of Sarbecoviruses.

microbiology↗

bioRxiv2022

Phenotypical Rescue of Bmp15 Deficiency by Mutation of Inhibin α (inha) Provides Novel Clues to How Bmp15 Controls Zebrafish Folliculogenesis

As an oocyte-specific growth factor, bone morphogenetic protein 15 (BMP15) plays a critical role in controlling folliculogenesis. However, the mechanism of BMP15 action remains elusive. Using zebrafish as the model, we created a bmp15 mutant using CRISPR/Cas9 and demonstrated that bmp15 deficiency caused a significant delay in follicle activation and puberty onset followed by complete arrest of follicle development at previtellogenic stage without yolk accumulation. The mutant females eventually underwent female-to-male sex reversal to become functional males, which was accompanied by a series of changes in secondary sexual characteristics. Interestingly, the blockade of folliculogenesis and sex reversal in bmp15 mutant could be rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic stage with yolk accumulation and the fish maintained their femaleness without sex reversal. Transcriptome analysis revealed up-regulation of pathways related to TGF-{beta} signaling and endocytosis in the double mutant follicles. Intriguingly, the expression of inhibin/activin {beta}Aa subunit (inhbaa) increased significantly in the double mutant ovary. Further knockout of inhbaa in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-) resulted in the loss of yolk granules again in the oocytes although the follicles could continue to grow beyond the size range of previtellogenic stage. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly in bmp15 single mutant females, returned to normal in the double mutant (bmp15-/-;inha-/-), but reduced again significantly in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-). E2 treatment could rescue the vitellogenic follicles in bmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation in bmp15-/-;inha-/- fish. In summary, the present study provided comprehensive genetic evidence for the interaction of bmp15 pathways and the activin-inhibin system in regulating folliculogenesis, in particular E2 production from the follicle, Vtg biosynthesis in the liver and its update by the developing oocytes.

developmental biology↗

bioRxiv2022

DeepRTAlign: toward accurate retention time alignment for large cohort mass spectrometry data analysis

Retention time (RT) alignment is one of the crucial steps in liquid chromatography-mass spectrometry (LC-MS)-based proteomic and metabolomic experiments, especially for large cohort studies, and it can be achieved using computational methods; the most popular methods are the warping function method and the direct matching method. However, the existing tools can hardly handle monotonic and non-monotonic RT shifts simultaneously. To overcome this, we developed a deep learning-based RT alignment tool, DeepRTAlign, for large cohort LC-MS data analysis. It first performs a coarse alignment by calculating the average time shift between any two samples and then uses RT and m/z as the main features to train its deep learning-based model. We demonstrate that DeepRTAlign has improved performances, especially when handling complex samples, by benchmarking it against current state-of-the-art approaches on 19 real-world proteomic and metabolomic datasets and the corresponding simulated datasets. Benchmarked on a dataset with known fold changes, the results showed that DeepRTAlign can improve the identification sensitivity of MS data without compromising the quantitative accuracy. Furthermore, using the MS features aligned by DeepRTAlign in a large cohort, we trained a classifier of 15 features to predict the early recurrence of hepatocellular carcinoma. The features were validated on an independent cohort using targeted proteomics with an AUC of 0.833. Being flexible and robust with four different feature extraction tools, DeepRTAlign provides an advanced solution to RT alignment in large cohort LC-MS data, which is currently one of the bottlenecks in proteomics and metabolomics research, especially for clinical applications.

bioinformatics↗

bioRxiv2022

The deacetylase dependent and independent role of HDAC3 in cardiomyopathy

BackgroundCardiomyopathy is a common disease of cardiac muscle that negatively affects cardiac function. HDAC3 commonly functions as co-repressor by removing acetyl moieties from histone tails. However, a deacetylase-independent role of HDAC3 has also been described. Cardiac deletion of HDAC3 causes reduced cardiac contractility accompanied by lipid accumulation. The molecular function of HDAC3 in cardiomyopathy remains unknown. We have used the powerful genetic tools in Drosophila to investigate the enzymatic and non-enzymatic roles of HDAC3 in cardiomyopathy. Methods and ResultsUsing the Drosophila heart model, we showed that cardiac-specific HDAC3 knockdown leads to prolonged systoles and reduced cardiac contractility. Immunohistochemistry revealed structural abnormalities characterized by myofiber disruption in HDAC3 knock down hearts. Cardiac-specific HDAC3 knockdown showed increased levels of whole body triglycerides and increased fibrosis. The introduction of deacetylase-dead HDAC3 mutant in HDAC3 KD background showed comparable results with wild type HDAC3 in aspects of contractility and Pericardin deposition. However, deacetylase-dead HDAC3 mutants failed to improve triglyceride accumulation. ConclusionsOur data indicate that HDAC3 plays a deacetylase-independent role in maintaining cardiac contractility and preventing Pericardin deposition as well as a deacetylase-dependent role to maintain triglyceride homestasis.

physiology↗

bioRxiv2022

Genetic Analysis of Activin/Inhibin β Subunits in Zebrafish Development and Reproduction

Activin and inhibin are both dimeric proteins sharing the same {beta} subunits that belong to the TGF-{beta} superfamily. They are well known for stimulating and inhibiting pituitary FSH secretion, respectively, in mammals. In addition, activin also acts as a mesoderm-inducing factor in frogs. However, their functions in development and reproduction of other species are poorly defined. In this study, we disrupted all three activin/inhibin {beta} subunits ({beta}Aa, inhbaa; {beta}Ab, inhbab; and {beta}B, inhbb) in zebrafish using CRISPR/Cas9. The loss of {beta}Aa/b but not {beta}B led to a high mortality rate in the post-hatching stage. Surprisingly, the expression of fshb but not lhb in the pituitary increased in the female {beta}A mutant together with aromatase (cyp19a1a) in the ovary. The single mutant of {beta}Aa/b showed normal folliculogenesis in young females; however, their double mutant (inhbaa-/-;inhbab-/-) showed delayed follicle activation, granulosa cell hypertrophy, stromal cell accumulation and tissue fibrosis. The ovary of inhbaa-/- deteriorated progressively after 180 dpf with reduced fecundity and the folliculogenesis ceased completely around 540 dpf. In addition, tumor- or cyst-like tissues started to appear in the inhbaa-/- ovary after about one year. In contrast to females, activin {beta}Aa/b mutant males showed normal spermatogenesis and fertility. As for activin {beta}B subunit, the inhbb-/- mutant exhibited normal folliculogenesis, spermatogenesis and fertility in both sexes; however, the fecundity of mutant females decreased dramatically at 270 dpf with accumulation of early follicles. In summary, the activin-inhibin system plays an indispensable role in fish reproduction, in particular folliculogenesis and ovarian homeostasis.

physiology↗

bioRxiv2022

High Resolution Imaging of the Osteogenic and Angiogenic Interface at the Site of Cranial Bone Defect Repair via Multiphoton Microscopy

The spatiotemporal blood vessel formation and specification at the osteogenic and angiogenic interface of cranial bone defect repair were examined utilizing a high-resolution multiphoton-based imaging platform in conjunction with advanced optical techniques that allow interrogation of the oxygen microenvironment and cellular energy metabolism in living animals. Our study demonstrates the dynamic changes of vessel types, i.e. arterial, venous and capillary vessel networks at the superior and dura periosteum of cranial bone defect, suggesting a differential coupling of the vessel type with osteoblast expansion and bone tissue deposition/remodeling during repair. Employing transgenic reporter mouse models that label distinct types of vessels at the site of repair, we further show that oxygen distributions in capillary vessels at the healing site are heterogeneous as well as time and location-dependent. The endothelial cells coupling to osteoblasts prefer glycolysis and are less sensitive to microenvironmental oxygen changes than osteoblasts. In comparison, osteoblasts utilize relatively more OxPhos and potentially consume more oxygen at the site of repair. Taken together, our study highlights the dynamics and functional significance of blood vessel types at the site of defect repair, opening up opportunities for further delineating the oxygen and metabolic microenvironment at the interface of bone tissue regeneration.

bioengineering↗

bioRxiv2022

Targeted multi-omic analysis of human skin tissue identifies alterations of conventional and unconventional T cells associated with burn injury

Burn injuries are a leading cause of unintentional injury, associated with a dysfunctional immune response and an increased risk of infections. Despite this, little is known about the role of T cells in human burn injury. In this study, we compared the activation and function of conventional T cells and unconventional T cell subsets in skin tissue from acute burn (within 7 days from initial injury) burn, late phase burn (beyond 7 days from initial injury), and non-burn patients. We compared T cell functionality by a combination of flow cytometry and a multi-omic single-cell approach with targeted transcriptomics and protein expression. We found a significantly lower proportion of CD8+ T cells in burn skin compared to non-burn skin, with CD4+ T cells making up the bulk of the T cell population. Both conventional and unconventional burn tissue T cells show significantly higher IFN-{gamma} and TNF- levels after stimulation than non-burn skin T cells. In sorted T cells, clustering showed that burn tissue had significantly higher expression of homing receptors CCR7, S1PR1, and SELL compared to non-burn skin. In unconventional T cells, including mucosal-associated invariant T (MAIT) and {gamma}{delta} T cells, we see significantly higher expression of cytotoxic molecules GZMB, PRF1, and GZMK. Multi-omics analysis of conventional T cells suggests a shift from tissue-resident T cells in non-burn tissue to a circulating T cell phenotype in burn tissue. In conclusion, by examining skin tissue from burn patients, our results suggest that T cells in burn tissue have a pro-inflammatory rather than a homeostatic tissue-resident phenotype, and that unconventional T cells have a higher cytotoxic capacity. Our findings have the potential to inform the development of novel treatment strategies for burns.

immunology↗

bioRxiv2022

Testis-specific H2BFWT disrupts nucleosome integrity through reductions of DNA-histone interactions

During spermatogenesis, multiple testis-specific histone variants are involved in the dynamic chromatin transitions. H2BFWT is a primate testis-specific H2B variant with hitherto unclear functions, and SNPs of H2BFWT are closely associated with male non-obstructive infertility. Here, we found that H2BFWT is preferentially localized in the sub-telomeric regions and the promoters of genes highly expressed in testis from differentiated spermatogonia to early spermatocytes. Cryo-EM structural analysis shows that H2BFWT nucleosomes are defined by weakened interactions between H2A-H2BFWT dimer and H4, and between histone octamer and DNA. Furthermore, one of its SNPs, H2BFWTH100R further destabilizes nucleosomes and increases the nucleosome unwrapping rate by interfering with the interaction with H4K91. Our results suggest that H2BFWT may be necessary for the regulation of spermatogenesis-related gene expression by decreasing transcriptional barriers, and that H2BFWTH100R overdrives its nucleosome-destabilizing effects which causes infertility.

biochemistry↗

bioRxiv2022

Starvation Response and RNA Polymerase Guide Antibiotic-induced Mutable Gambler Cells

Antibiotics can induce mutations that cause antibiotic resistance, triggered by stress responses. The identities of the stress-response activators reveal environmental cues that elicit mutagenesis, and are weak links in mutagenesis networks, inhibition of which could slow evolution of resistance during antibiotic therapies. Despite pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin (stress)-induced mutagenesis. We show its role in transient differentiation of a mutable "gambler" cell subpopulation. Stringent activity is induced by reactive oxygen and upregulates two small (s)RNAs that induce the general stress response, which allows mutagenic DNA-break repair. Surprisingly, RNA polymerase and response-activator nucleotide (p)ppGpp promote ciprofloxacin-induced DNA-damage signaling. We propose a model for their regulation of DNA-damage processing in transcribed regions. The data demonstrate a critical node in ciprofloxacin-induced mutagenesis, imply RNA-polymerase regulation of DNA-break repair, and identify promising targets for resistance-resisting drugs.

microbiology↗

bioRxiv2022

Loss of growth differentiation factor 9 causes an arrest of early folliculogenesis in zebrafish - a novel insight into its action mechanism

Growth differentiation factor 9 (GDF9) is the best characterized growth factor released by the oocyte; however, most information about GDF9 functions comes from studies in the mouse model. In this study, we created a mutant for Gdf9 gene (gdf9-/-) in zebrafish using TALEN approach. The loss of Gdf9 caused a complete arrest of follicle development at primary growth (PG) stage. These follicles eventually degenerated, and all mutant females gradually changed to males through sex reversal, which could be prevented by mutation of the male-promoting gene dmrt1. Interestingly, the phenotypes of gdf9-/- could be rescued by mutation of inhibin (inha-/-) but not estradiol, suggesting a potential role for the activin-inhibin system in Gdf9 actions. In gdf9 null follicles, activin {beta}Aa (inhbaa) expression decreased dramatically; however, its expression rebounded in the double mutant (gdf9-/-;inha-/-). These results clearly indicate that although endocrine hormones such as follicle-stimulating hormone (FSH) are important for folliculogenesis, the activation of PG follicles requires intrinsic factors from the oocyte, such as Gdf9, which in turn works on the neighboring follicle cells to trigger follicle activation, probably via activins.

developmental biology↗

bioRxiv2022

Longitudinal single-cell transcriptomics reveals distinct patterns of recurrence in acute myeloid leukemia

The heterogeneity and evolution of AML blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. To gain insight into the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs, we employed whole-exome sequencing and single-cell RNA-seq to longitudinally profile two t(8;21) (AML1-ETO = RUNX1-RUNX1T1), and four FLT3-ITD AML cases. The single cell RNA data underpinned the tumor heterogeneity amongst patient blasts. The Dx-Re transcriptomes of high risk FLT3-ITD pairs formed a continuum from extensively changed in the absence of significantly mutational changes in AML-associated genes to rather similar Dx-Re pair of an intermediate risk FLT3-ITD. In one high risk FLT3-ITD pair, a pathway switched from an AP-1 regulated network in Dx to mTOR signaling in Re. The distinct AML1-ETO pairs comprise clusters that share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells probably evolved from the Dx LSC-like cells. In summary, our study revealed a continuum from drastic transcriptional changes to extensive similarities between respective Dx-Re pairs that are poorly explained by the well-established model of clonal evolution. Our results suggest alternative and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.

cancer biology↗

bioRxiv2021

A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein

Cryo-electron microscopy (cryo-EM) has become the most powerful tool to resolve the high-resolution structures of biomacromolecules in solution. However, the air-water interface induced preferred orientation, dissociation or denaturation of biomacromolecules during cryo-vitrification is still a major limitation factor for many specimens. To solve this bottleneck, we developed a new type of cryo-EM support film using the 2D crystal of hydrophobin I (HFBI) protein. The HFBI-film utilizes its hydrophilic side to adsorb protein particles via electrostatic interactions and keep air-water interface away, allowing thin-enough ice and high-quality data collection. The particle orientation distribution can be optimized by changing the buffer pH. We, for the first time, solved the cryo-EM structure of catalase (2.28-[A]) that exhibited strong preferred orientation using conventional cryo-vitrification protocol. We further proved the HFBI-film is suitable to solve the high-resolution structures of small proteins including aldolase (150 kDa, 3.34-[A]) and hemoglobin (64 kDa, 3.6-[A]). Our work implied that the HFBI-film will have a wide application in the future to increase the successful rate and efficiency of cryo-EM.

biophysics↗

bioRxiv2020

Single-cell RNA-sequencing of peripheral neuroblastic tumors reveals an aggressive transitional cell state at the junction of an adrenergic-mesenchymal transdifferentiation trajectory

Peripheral neuroblastic tumors (PNTs) are the most common extracranial solid tumors in early childhood. They represent a spectrum of neural crest derived tumors including neuroblastoma, ganglioneuroblastoma and ganglioneuroma. PNTs exhibit heterogeneity due to interconverting malignant cell states described as adrenergic/nor-adrenergic or mesenchymal/neural crest cell in origin. The factors determining individual patient levels of tumor heterogeneity, their impact on the malignant phenotype, and the presence of other cell states are unknown. Here, single-cell RNA-sequencing analysis of 4267 cells from 7 PNTs demonstrated extensive transcriptomic heterogeneity. Trajectory modelling showed that malignant neuroblasts move between adrenergic and mesenchymal cell states via a novel state that we termed a "transitional" phenotype. Transitional cells are characterized by gene expression programs linked to a sympathoadrenal development, and aggressive tumor phenotypes such as rapid proliferation and tumor dissemination. Among primary bulk tumor patient cohorts, high expression of the transitional gene signature was highly predictive of poor prognosis when compared to adrenergic and mesenchymal expression patterns. High transitional gene expression in neuroblastoma cell lines identified a similar transitional H3K27-acetylation super-enhancer landscape, supporting the concept that PNTs have phenotypic plasticity and transdifferentiation capacity. Additionally, examination of PNT microenvironments, found that neuroblastomas contained low immune cell infiltration, high levels of non-inflammatory macrophages, and low cytotoxic T lymphocyte levels compared with more benign PNT subtypes. Modeling of cell-cell signaling in the tumor microenvironment predicted specific paracrine effects toward the various subtypes of malignant cells, suggesting further cell-extrinsic influences on malignant cell phenotype. Collectively, our study reveals the presence of a previously unrecognized transitional cell state with high malignant potential and an immune cell architecture which serve both as potential biomarkers and therapeutic targets.

cancer biology↗

bioRxiv2020

TWIRLS, an automated topic-wise inference method based on massive literature, suggests a possible mechanism via ACE2 for the pathological changes in the human host after coronavirus infection

Faced with the current large-scale public health emergency, collecting, sorting, and analyzing biomedical information related to the "coronavirus" should be done as quickly as possible to gain a global perspective, which is a basic requirement for strengthening epidemic control capacity. However, for human researchers studying the viruses and the hosts, the vast amount of information available cannot be processed effectively and in a timely manner, particularly when the scientific understanding may be limited, which can further lower the information processing efficiency. We present TWIRLS, a method that can automatically acquire, organize, and classify information. Additionally, independent functional data sources can be added to build an inference system using a machine-based approach, which can provide relevant knowledge to help human researchers quickly establish subject cognition and to make more effective decisions. TWIRLS can automatically analyze more than three million words in more than 14,000 literature articles in only 4 hours. Combining with generalized gene interaction databases creates a data interface that can help researchers to further analyze the information. Using the TWIRLS system, we found that an important regulatory factor angiotensin-converting enzyme 2 (ACE2) may be involved in the host pathological changes on binding to the coronavirus after infection. After triggering functional changes in ACE2/AT2R, an imbalance in the steady-state cytokine regulatory axis involving the Renin-Angiotensin System and IP-10 leads to a cytokine storm.

bioinformatics↗

bioRxiv2019

Stress Resistance Screen in a Human Primary Cell Line Identifies Small Molecules that Affect Aging Pathways and Extend C. elegans Lifespan

Increased resistance to environmental stress at the cellular level is correlated with the longevity of long-lived mutants and wild-animal species. Moreover, in experimental organisms, screens for increased stress resistance have yielded mutants that are long-lived. To find entry points for small molecules that might extend healthy longevity in humans, we screened [~]100,000 small molecules in a human primary-fibroblast cell line and identified a set that increased oxidative-stress resistance. Some of the hits fell into structurally-related chemical groups, suggesting that they may act on common targets. Two small molecules increased C. elegans stress resistance, and at least 9 extended their lifespan by [~]10-50%. Thus, screening for increased stress resistance in human cells can enrich for compounds with promising pro-longevity effects. Further characterization of these compounds, including a chalcone that promoted stress resistance independently of NRF2, may elucidate new ways to extend healthy human lifespan.

genetics↗

bioRxiv2019

Molecular mechanism of mitochondrial phosphatidate transfer by Ups1/Mdm35

Cardiolipin plays many important roles for mitochondrial physiological function and is synthesized from phosphatidic acid (PA) at inner mitochondrial membrane (IMM). PA synthesized from endoplasmic reticulum needs to transfer to IMM via outer mitochondrial membrane (OMM). The transfer of PA between IMM and OMM is mediated by Ups1/Mdm35 protein family. Although there are many structures of this family available, the detailed molecular mechanism of how PA is transferred between membranes is yet unknown. Here, we report another crystal structures of Ups1/Mdm35 in the authentic monomeric apo state and the DHPA bound state. By combining subsequent all-atom molecular dynamics simulations, extensive structural comparisons and biophysical assays, we discovered the conformational changes of Ups1/Mdm35, identified key structural elements and residues during membrane binding and PA entry. We found the monomeric Ups1 on membrane is an important transit for the success of PA transfer, and the equilibrium between monomeric Ups1 and Ups1/Mdm35 complex on membrane affects the PA transfer rate and can be regulated by many factors including environmental pH.

biochemistry↗