Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 235 records · Page 13Linked to original sources

bioRxiv2024

Causal Brain Network Evaluates Surgical Outcomes in Patients with Drug-Resistant Epilepsy: A Retrospective Comparative Study

Network neuroscience has greatly facilitated epilepsy studies; meanwhile, drug-resistant epilepsy (DRE) is increasingly recognized as a brain network disorder. Unfortunately, surgical success rates in patients with DRE are still very limited, varying 30% [~] 70%. At present, there is almost no systematic exploration of intracranial electrophysiological brain network closely related to surgical outcomes, and it is not clear which brain network methodologies can effectively promote DRE precision medicine. In this retrospective comparative study, we included multicenter datasets, containing electrocorticogram (ECoG) data from 17 DRE patients with 55 seizures. Ictal ECoG within clinically-annotated epileptogenic zone (EZ) and non-epileptogenic zone (NEZ) were separately computed using six different algorithms to construct causal brain networks. All the brain network results were divided into two groups, successful and failed surgery. Statistical results based on the Mann-Whitney-U-test show that: causal connectivity of -frequency band (8 [~] 13 Hz) in EZ calculated by convergent cross mapping (CCM) gains the most significant differences between the surgical success and failure groups, with a P value of 7.85e-08 and Cohens d effect size of 0.77. CCM-defined EZ brain network can also distinguish the successful and failed surgeries considering clinical covariates (clinical centers, DRE types) with P < 0. 001. Based on the brain network features, machine learning models are established to predict the surgical outcomes. Among them, SVM classifier with Gaussian kernel function and Bayesian Optimization demonstrates the highest average accuracy of 84.48% through 5-fold cross validation, further indicating that the CCM-defined EZ brain network is a reliable biomarker for predicting DREs surgical outcomes.

neuroscience↗

bioRxiv2024

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both basal and induced mitophagy. Disruption of the phosphatase activity of PPTC7 is not required for BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-NIX/BNIP3 and PPTC7-FBXL4 interactions and their disruption interferes with both NIX/BNIP3 degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts NIX/BNIP3-induced mitophagy.

cell biology↗

bioRxiv2024

Characterization of the SARS-CoV-2 BA.5 Variants in H11-K18-hACE2 Hamsters

This study aims to comprehensively characterize the SARS-CoV-2 BA.5 variants using K18 hACE2 transgenic mice and golden hamsters as model organisms. Previous research on SARS-CoV-2 has utilized both mouse and hamster models, leading to conflicting results concerning the viruss lethality. In our study, the finding suggests that H11-K18 hACE2 golden hamsters closely mimic the disease progression observed in human COVID-19 cases caused by BA.5 variants, demonstrating consistent severity and symptoms comparable to severe infections. Additionally, hamsters exhibit heightened respiratory viral replication, accurately reflecting the clinical viral kinetics observed in humans. The study emphasizes the critical importance of selecting an appropriate animal model for SARS-CoV-2 research, while also providing robust support for the hypothesis that BA.5 variants contribute to fatal outcomes in COVID-19 cases. These findings highlight the pivotal role of the golden hamster model in advancing our understanding of the pathogenic mechanisms underlying SARS-CoV-2 variants, as well as in the development of targeted therapeutic strategies. Significance StatementOur research work explores groundbreaking insights that could reshape our understanding of COVID-19 and pave the way for targeted therapies. We use golden hamster models to express the possibility of different animal models could contribute to human diseases. We hope this finding could clarify some conflicts existed, and help further development of medication for COVID.

immunology↗

bioRxiv2024

Visualizing the M2 muscarinic acetylcholine receptor activation regulated by aromatic ring dynamics

A detailed molecular understanding of the G-protein coupled receptor (GPCR) activation mechanism is crucial for rational drug design. Despite the growing number of GPCR structures being resolved in the inactive and activated states, the detailed molecular mechanism of how receptors transits from the inactive towards the active one upon agonist binding remains to be further understood. Herein, we performed comprehensive atomic-level simulations of the M2 muscarinic receptor (M2R) to determine how ligand binding modulates the receptor conformational dynamics. The results reveal that aromatic residue dynamics are closely associated with receptor activation. Binding of antagonists or agonists differentially alters the interacting patterns of critical aromatic residues, stabilizing them into distinct conformations. In addition, we found that the change of interaction and dynamics of the W4006.48-F3966.44 pair at the transmembrane core plays an essential role in the structural transition of M2R from the inactive state into an active-like state induced by binding of the supra-physiological agonist iperoxo. Moreover, we found that the sidechain dynamics of Y2065.58 is important in modulating the conformation of the intracellular cavity. Our work highlights the role of protein conformational dynamics in M2R activation, and provides new insights for future drug designs.

biochemistry↗

bioRxiv2024

DOPAMINE D4 RECEPTOR DOWN-REGULATES RENAL SODIUM CHLORIDE COTRANSPORTER VIA UBIQUITINATION-ASSOCIATED LYSOSOME DEGRADATION

BackgroundThe thiazide-sensitive sodium chloride cotransporter (NCC) is the major apical sodium transporter located in the mammalian renal distal convoluted tubule (DCT). The amount of sodium reabsorbed in the DCT through NCC plays an important role in the regulation of extracellular fluid volume and blood pressure. Dopamine and its receptors constitute a renal antihypertensive system in mammals. The disruption of Drd4 in mice causes kidney-related hypertension. However, the pathogenesis of D4R-deficiency associated hypertension is not well documented. MethodWe assessed the effects of D4R on NCC protein abundances and activities of DCT in mice with renal or global Drd4-deficiencies and expressing human D4.7 variant and in cultured mouse DCT cells, and explored the molecular mechanism. ResultsNCC inhibitor hydrochlorothiazide enhanced the natriuresis in Drd4-/- mice. Renal NCC protein was greater while ubiquitination of NCC was less in Drd4-/- than Drd4+/+ mice. Silencing of D4R in cultured mouse DCT cells increased NCC protein but decreased NCC ubiquitination. D4R agonist had opposite effects that were blocked by the antagonist. In mouse kidneys and DCT cells D4R and NCC colocalized and co-immunoprecipitated. Moreover, D4R-agonist promoted the binding between the two proteins demonstrated by fluorescence resonance energy transfer. D4R agonism internalized NCC, decreased NCC in the plasma membrane, increased NCC in lysosomes and reduced NCC-dependent-intracellular-sodium transport. The lysosomal inhibitor chloroquine prevented the D4R-induced NCC-reduction. A shortened NCC half-life was suggested by its decay under cycloheximide-chase. Ubiquitin-specific-protease 48 (USP48, a deubiquitinating enzyme) was increased in the kidneys and cells with Drd4-deficiency while D4R stimulation decreased it in vitro and reduction of USP48 with siRNA decreased NCC expression. The mice carrying human D4.7 variant or with renal reduction of D4R developed hypertension with increased NCC. ConclusionOur data demonstrates that D4R downregulates NCC by promoting USP48-associated deubiquitination and subsequent internalization, lysosome relocation and degradation.

physiology↗

bioRxiv2024

Ketone body 3-hydroxybutyrate alleviates CCl4-induced liver fibrosis in mice through anti-inflammatory effects

3-Hydroxybutyrate (3HB) is an important metabolite and regulatory molecule produced in liver. Previous studies have shown that 3HB could be beneficial to many diseases, including brain diseases, diabetes, and most importantly, inflammation and liver related injury. Therefore, the effect of 3HB on liver fibrosis, one key step of liver diseases which proved to be reversible, is urgent to explore. In this study, the CCl4-induced mouse model of liver fibrosis has been successfully constructed and treated by 3HB. The results demonstrate that 3HB could alleviate CCl4-induced liver injury and inflammation in mice, decrease the accumulation of collagen, the expression of pro-fibrotic genes as well as inflammatory factors, and finally the degree of liver fibrosis. The transcriptome data recovers that the anti-fibrotic effect of 3HB might be exerted through several ways, such as regulating mitochondrial function, reducing oxidative stress and p53 signaling pathways, proposing a safe and relatively fast possibility for the treatment of liver fibrosis.

biochemistry↗

bioRxiv2024

Mechanism and regulation of cargo entry into the Commander recycling pathway

Commander is a multiprotein complex that orchestrates endosomal recycling of diverse integral cargo proteins and in humans is required for normal skeletal, brain, kidney, and cardiovascular development. While the structure of this complex has recently been described, the central question of how cargo proteins are selected for entry into the Commander recycling pathway remains unclear. Here using recombinant protein reconstitution and in silico predictions we identify the evolutionary conserved mechanism through which the unstructured carboxy-terminal tail of the integral protein adaptor sorting nexin-17 (SNX17) directly binds to the Retriever sub-complex of Commander. SNX17 adopts an autoinhibited conformation where its carboxy-terminal tail occupies the cargo binding groove. Competitive cargo binding overcomes this autoinhibition, promoting SNX17 endosomal residency and the release of the carboxy tail for Retriever association. Using molecular cell biology and high-resolution microscopy, we establish the central importance of SNX17-Retriever association in the handover of integrin and lipoprotein receptor cargoes into pre-existing endosomal retrieval sub-domains for entry into the recycling pathway. In describing the principal mechanism of cargo entry into the Commander recycling pathway we provide key insight into the function and regulation of this evolutionary conserved sorting complex.

cell biology↗

bioRxiv2024

Targeting transcription factors through an IMiD independent zinc finger domain

Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/574032v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@b8e4e8org.highwire.dtl.DTLVardef@bc6f01org.highwire.dtl.DTLVardef@11bbe85org.highwire.dtl.DTLVardef@791cb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Optimal distance metrics for single-cell RNA-seq populations

In single-cell data workflows and modeling, distance metrics are commonly used in loss functions, model evaluation, and subpopulation analysis. However, these metrics behave differently depending on the source of variation, conditions and subpopulations in single-cell expression profiles due to data sparsity and high dimensionality. Thus, the metrics used for downstream tasks in this domain should be carefully selected. We establish a set of benchmarks with three evaluation measures, capturing desirable facets of absolute and relative distance behavior. Based on seven datasets using perturbation as ground truth, we evaluated 16 distance metrics applied to scRNA-seq data and demonstrated their application to three use cases. We find that linear metrics such as mean squared error (MSE) performed best across our three evaluation criteria. Therefore, we recommend the use of MSE for comparing single-cell RNA-seq populations and evaluating gene expression prediction models.

bioinformatics↗

bioRxiv2023

Branched-chain keto acids promote an immune-suppressive and neurodegenerative microenvironment in leptomeningeal disease

Leptomeningeal disease (LMD) occurs when tumors seed into the leptomeningeal space and cerebrospinal fluid (CSF), leading to severe neurological deterioration and poor survival outcomes. We utilized comprehensive multi-omics analyses of CSF from patients with lymphoma LMD to demonstrate an immunosuppressive cellular microenvironment and identified dysregulations in proteins and lipids indicating neurodegenerative processes. Strikingly, we found a significant accumulation of toxic branched-chain keto acids (BCKA) in the CSF of patients with LMD. The BCKA accumulation was found to be a pan-cancer occurrence, evident in lymphoma, breast cancer, and melanoma LMD patients. Functionally, BCKA disrupted the viability and function of endogenous T lymphocytes, chimeric antigen receptor (CAR) T cells, neurons, and meningeal cells. Treatment of LMD mice with BCKA-reducing sodium phenylbutyrate significantly improved neurological function, survival outcomes, and efficacy of anti-CD19 CAR T cell therapy. This is the first report of BCKA accumulation in LMD and provides preclinical evidence that targeting these toxic metabolites improves outcomes.

cancer biology↗

bioRxiv2023

Action-outcome based flexible behavior requires medial prefrontal cortex lead and its enhanced functional connectivity with dorsomedial striatum

Cognitive flexibility plays a key role in ensuring an individuals survival, and its deficit is a key symptom in many mental conditions and neurodegenerative diseases. The prefrontal cortex and striatum are both essential to cognitive flexibility. However, how the prefrontal cortex and striatum communicate with each other to enable flexible decision-making is not well understood. Competing theories are raised, debating on which structure among these two leads the role in detecting and representing the new circumstances for a change, giving largely opposing predictions on neural activities in the prefrontal cortex and striatum during flexible behavior. To address this question, we trained head-restrained mice to perform an action-outcome based dynamic foraging task and simultaneously recorded single-neuron activities in the medial prefrontal cortex (mPFC) and dorsomedial striatum (DMS). In this task, the animal chooses one of two actions to obtain reward. The animal is guided only by previous reward outcomes. We report that mPFC but not DMS activity stores information about prior reward history. A large fraction of both mPFC and DMS neurons activity represents the difference in reward probability between two alternative options, namely the perceived reward probability difference (PRPD), a key decision variable that prescribes which subsequent choice to make. We find that mPFC neural activities track the change of PRPD earlier and faster than those in the DMS, and functional connectivity between mPFC and DMS increases with reducing overall reward proportion.

neuroscience↗

bioRxiv2023

Modulation of Salmonella virulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex.

The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterise the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis, that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed between the five subspecies of Salmonella enterica, is found in many clinically-relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. Translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely {beta}-2-syntrophin, utrophin/ dystrophin, -catulin, -dystrobrevin and {beta}-dystrobrevin. The interaction between SseM and {beta}-2-syntrophin and -dystrobrevin was verified in S. Typhimurium-infected cells and relied on the PDZ domain of {beta}-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A {Delta}sseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates systemic growth of bacteria in mice. ImportanceIn Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells that are required for Salmonella virulence. The identification and functional characterisation of SPI-2 injectisome effectors advances our understanding of the interplay between Salmonella and its host(s). Using an optimised method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal (iNTS) Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector - SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a PDZ-binding motif that is essential for interaction with the PDZ-containing host protein {beta}-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.

microbiology↗

bioRxiv2023

Expansion of human hematopoietic stem cells by inhibiting translation

Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a [~]5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. These culture conditions maintain adult HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies. SignificanceO_LIWe report ex vivo expansion of human hematopoietic stem cells from umbilical cord blood using low cytokines and small molecule inhibitors of GSK-3, mTORC1, and the translation initiation factor eIF4E. C_LIO_LIOur method maintains human HSCs ex vivo after gene editing with conventional CRISPR/Cas9, lipid nanoparticle delivery, and base editing approaches, overcoming a major obstacle to gene editing for inherited blood disorders. C_LI

cell biology↗

bioRxiv2023

dFLASH; dual FLuorescent transcription factor Activity Sensor for Histone integrated live-cell reporting and high-content screening

Live-cell reporting of regulated transcription factor (TF) activity has a wide variety of applications in synthetic biology, drug discovery, and functional genomics. As a result, there is high value in the generation of versatile, sensitive, robust systems that can function across a range of cell types and be adapted toward diverse TF classes. Here we present the dual FLuorescent transcription factor Activity Sensor for Histone integrated live-cell reporting (dFLASH), a modular sensor for TF activity that can be readily integrated into cellular genomes. We demonstrate readily modified dFLASH platforms that homogenously, robustly, and specifically sense regulation of endogenous Hypoxia Inducible Factor (HIF) and Progesterone receptor (PGR) activities, as well as regulated coactivator recruitment to a synthetic DNA-Binding Domain-Activator Domain fusion proteins. The dual-colour nuclear fluorescence produced normalised dynamic live-cell TF activity sensing with facile generation of high-content screening lines, strong signal:noise ratios and reproducible screening capabilities (Z = 0.68-0.74). Finally, we demonstrate the utility of this platform for functional genomics applications by using CRISPRoff to modulate the HIF regulatory pathway, and for drug screening by using high content imaging in a bimodal design to isolate activators and inhibitors of the HIF pathway from a [~]1600 natural product library.

synthetic biology↗

bioRxiv2023

Unveiling unique microbial nitrogen cycling and novel nitrification drivers in coastal Antarctica

Although microbial nitrogen (N) cycling plays a pivotal role in Antarctic ecosystems, its underlying mechanisms are largely uncharted. In this study, we unravel the biological origin of nitrate via triple oxygen isotopic composition analysis and systematically profile functional N-cycling genes within soil and lake sediment samples from the ice-free areas of East Antarctica. We successfully reconstruct 1,968 metagenome-assembled genomes (MAGs) spanning 29 microbial phyla, enabling the analysis of the presence or absence of 52 diverse metabolic marker genes. Consistent with quantitative data, our metagenomic analyses confirm the active processes of microbial nitrogen fixation, nitrification, and denitrification. We find no detectable anaerobic ammonium oxidation (anammox) processes, underscoring a unique microbial N-cycling dynamic in the region. Notably, we identify the predominance of complete ammonia-oxidizing (comammox) Nitrospira, a recently discovered bacterial guild capable of performing the entire nitrification process within a single organism. Further genomic investigations reveal their adaptive strategies in the Antarctic environment. These strategies likely involve the synthesis of trehalose to counteract cold stress, high substrate affinity to efficiently utilize available resources, and alternative metabolic pathways to adapt to nutrient-scarce conditions. Their significant role in the nitrification process is validated through 13C-DNA-based stable isotope probing (DNA-SIP). This research provides a comprehensive illustration of nitrifications crucial contribution to the nitrogen budget in coastal Antarctica, highlighting comammox Nitrospira clade B as a novel nitrifying agent and shedding new light on the complex biogeochemical processes of nitrogen cycling in coastal Antarctica.

microbiology↗

bioRxiv2023

An NMR-guided approach for identifying co-factors in boosting the Pfu DNA polymerase

With rapid developments of emerging technologies like synthetic biology, the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly. Thus, rational optimization of the performance of DNA polymerase is of great interest. Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique used for studying protein structure and dynamics. It provides the atomic resolution information of enzymes under its functional solution environment to reveal the active sites (hot spots) of the enzyme, which could be further used for optimizing the performance of enzymes. In our previous work, we identified hot spot residues of Pfu DNA polymerase (Pfu pol). We aim to employ these binding hot spots to screen for co-factors of Pfu pol, particularly targeting those molecules exhibiting weak intermolecular interactions. To validate this concept, we first demonstrated the feasibility of utilizing hot spot residues as screening probes for auxiliary factors by employing the well-characterized Tween-20 as a model system. Employing these hot spots as probes, two new co-factors, the heat shock protein TkHSP20 from Thermococcus Kodakaraensis and the chemical chaperone L-arginine, are identified to interact with Pfu pol to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu pol. This NMR-based approach requires no prior assignment information of target enzymes, guiding the rational exploration of novel co-factors for Pfu pol. Moreover, our approach is not dependent on structural data or bioinformatics. Therefore, it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.

molecular biology↗

bioRxiv2023

Transcriptional coupling of telomeric retrotransposons with the cell cycle

Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in Drosophila, these are HeT-A, TART, and TAHRE. Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. Here we report the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in Drosophila. Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division.

cell biology↗

bioRxiv2023

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexan Cryptosporidium parvum

The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised/weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here we report a new micronemal protein in C. parvum, namely thrombospondin repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by selective kinesin-5 inhibitors, SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize {gamma}-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and biochemically validated for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells. Importance Cryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding on the composition, secretory pathways and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely CpTSP4, that is discharged during excystation, gliding and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion and heparin/heparan sulfate is one of the ligands for this micronemal protein.

microbiology↗