Biology subjects

Nie, L.

Publications and source records attributed to Nie, L..

32 records · Page 2Linked to original sources

bioRxiv2023

DCBLD1 modulates angiogenesis by regulating of the VEGFR-2 endocytosis in endothelial cells

ObjectiveUnwanted angiogenesis is involved in the progression of various malignant tumors and cardiovascular diseases, and the factors that regulate angiogenesis are potential therapeutic targets. We tested the hypothesis that DCBLD1 (Discoidin, CUB, and LCCL domain-containing protein 1) is a co-receptor of VEGFR-2 and modulates angiogenesis in endothelial cells(ECs). Approach and ResultsA carotid artery ligation model and retinal angiogenesis assay were used to study angiogenesis using globe knockout or EC-specific conditional DCBLD1 knockout mice in vivo. Immunoblotting, immunofluorescence staining, plasma-membrane subfraction isolation, Co-immunoprecipitation and mass-spectrum assay were performed to clarify the molecular mechanisms.Loss of DCBLD1 impaired VEGF response and inhibited VEGF-induced EC proliferation and migration. DCBLD1 deletion interfered with adult and developmental angiogenesis. Mechanistically, DCBLD1 bound to VEGFR-2 and regulated the formation of VEGFR-2 complex with negative regulators: protein tyrosine phosphatases, E3 ubiquitin ligases(Nedd4 and c-Cbl), and also DCBLD1 knockdown promoted lysosome-mediated VEGFR-2 degradation in ECs. ConclusionsThese findings demonstrated the essential role of endothelial DCBLD1 in regulating VEGF signaling and provided evidence that DCBLD1 promotes VEGF-induced angiogenesis by limiting the dephosphorylation, ubiquitination, and lysosome degradation after VEGFR-2 endocytosis. We proposed that endothelial DCBLD1 is a potential therapeutic target for ischemic cardiovascular diseases by the modulation of angiogenesis through regulating of the VEGFR-2 endocytosis.

molecular biology↗

bioRxiv2023

Shin in the eye: Au-CS@FNDs as contact lens additives for blocking UV irradiation, bacterial keratitis, and corneal neovascularizationtherapies

Ophthalmic disease treatment remains a significant problem globally, resulting in poor vision and blindness. UV irradiation and bacterial infection may cause severe damage to the corneal, leading to vision loss within a few days. Corneal neovascular abnormally growth that bock lights reach to eyes, causes low vision. Thus it requires urgent and efficient clinical treatment. Contact lenses play an essential role in treating ophthalmic issues. In this work, we synthesized the chitosan-stabilized Au nanoparticles with a simple method. The Au nanoparticles were further physically adsorbed with negatively charged fluorescent nanodiamonds, yielding Au-CS@FNDs. These Au-CS@FNDs particles were proven with excellent UV adsorption, antibacterial properties, and photothermal conversation ability. Furthermore, we embedded Au-CS@FNDs particles into contact lenses to prevent corneal damage from UV light and bacterial infection. Moreover, the Au-CS@FNDs embedded contact lenses were used to inhibit the neovascularization in the Human Vascular Endothelial Cells via the photodynamic effect of Au nanoparticles. To the best of our knowledge, it is the first time that gold and diamond nanoparticles were used as additives in contact lenses, aiming at clinically corneal neovascular. Our results suggest that the controllable photothermal effect of Au-CS@FNDs embedded contact lenses may provide a unique way to intervene the neovascular-induced vision loss. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/536356v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1b94b65org.highwire.dtl.DTLVardef@4f598org.highwire.dtl.DTLVardef@198058forg.highwire.dtl.DTLVardef@1950cd3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Label-free differential imaging of cellular components in mouse brain tissue by wide-band photoacoustic microscopy

Mapping diverse cellular components with high spatial resolution is important to interrogate biological systems and study disease pathogenesis. Conventional optical imaging techniques for mapping biomolecular profiles with differential staining and labeling methods are cumbersome. Different types of cellular components exhibit distinctive characteristic absorption spectra across a wide wavelength range. By virtue of this property, a lab-made wide-band optical-resolution photoacoustic microscopy (wbOR-PAM) system, which covers wavelengths from the ultraviolet and visible to the shortwave infrared regions, was designed and developed to capture multiple cellular components in 300-m-thick brain slices at nine different wavelengths without repetitive staining and complicated processing. This wbOR-PAM system provides abundant spectral information. A reflective objective lens with an infinite conjugate design was applied to focus laser beams with different wavelengths, avoiding chromatic aberration. The molecular components of complex brain slices were probed without labeling. The findings of the present study demonstrated a distinctive absorption of phospholipids, a major component of the cell membrane, brain, and nervous system, at 1690 nm and revealed their precise distribution with microscopic resolution in a mouse brain, for the first time. This novel imaging modality provides a new opportunity to investigate important biomolecular components without either labeling or lengthy specimen processing, thus, laying the groundwork for revealing cellular mechanisms involved in disease pathogenesis.

bioengineering↗

bioRxiv2022

Revealing the molecular mechanisms underlying neural transmission of female pheromone signals in the male antennae of Antheraea pernyi by integrative proteomics and metabolomics analysis

Detection of sex pheromones of insects relies on the antennae. The female pheromone signal transmission in the male antennae ultimately initiates the courtship and mating behaviors of males. To investigate the proteins and metabolites involved in this neural transduction, the study adopted integrative proteomics and metabolomics analysis including tandem mass tag (TMT) proteomic quantification and liquid chromatography tandem mass spectrometry (LC/MS)-based metabolomics for comparing proteomic and metabolic changes between the antennae of male moths following stimulation by females and the non-stimulated males of A. pernyi. A total of 92 differentially expressed proteins (DEPs) containing 52 up-regulated and 40 down-regulated proteins and 545 differentially expressed metabolites (DEMs) including 218 up- and 327 down-regulated metabolites were identified from the antennae of female-stimulated male moths based on the proteome and metabolome data, respectively. GO enrichment analysis showed that 45 DEPs could be enriched into different GO terms on different levels. COG analysis indicated that 61 DEPs were assigned to 20 functional categories. The 160 DEMs respectively fell into 11 and 44 classes at SuperClass and Class levels based on HMDB annotation. KEGG pathway enrichment analysis indicated that totally 43 DEMs were enriched into 6, 27, and 87 pathways on level 1, 2, and 3, respectively. A number of DEPs and DEMs related to neural transmission of female pheromone signals in the male antennae of A. pernyi were screened, including tyrosine hydroxylase, cryptochrome-1, tachykinin, arylalkylamine N-acetyltransferase, cadherin-23, glutathione S-transferase delta 3, tyramine, tryptamine, n-oleoyl dopamine, n-stearoyl dopamine, and n-stearoyl tyrosine. We concluded that the altered expression levels of those proteins or metabolites were involved in regulating the neuron activity for enhanced transmission of neural impulses and continuous perception, reception, and transduction of female pheromone signals. Our findings yielded novel insights into the potential molecular mechanisms in the antennae of male A. pernyi responding to female attraction.

molecular biology↗

bioRxiv2021

PRE-CLINICAL IMMUNE RESPONSE AND SAFETY EVALUATION OF THE PROTEIN SUBUNIT VACCINE NANOCOVAX FOR COVID-19

The Coronavirus disease-2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has become a dire global health concern. The development of vaccines with high immunogenicity and safety is crucial for control of the global COVID-19 pandemic and prevention of further illness and fatalities. Here, we report development of SARS-CoV-2 vaccine candidate, Nanocovax, based on recombinant protein production of the extracellular (soluble) portion of the S protein of SARS-CoV-2. The results showed that Nanocovax induced high levels of S protein-specific IgG, as well neutralizing antibody in three animal models including Balb/C mice, Syrian hamsters, and non-human primate (Macaca leonina). In addition, the viral challenge study using the hamster model showed that Nanocovax protected the upper respiratory tract from SARS-CoV-2 infection. No adverse effects were induced by Nanocovax in swiss mice (Musmusculus var. Albino), Rats (Rattus norvegicus), and New Zealand rabbits. These pre-clinical results indicated that Nanocovax is safe and effective.

immunology↗

bioRxiv2021

Comprehensive analysis of the host-virus interactome of SARS-CoV-2

Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.

molecular biology↗

bioRxiv2020

The structural basis of fatty acid elongation by the ELOVL elongases

Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinsons, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 [A] long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy.

biochemistry↗

bioRxiv2020

Cleavage Site Specificity for Processing of Farnesylated Prelamin A by the Zinc Metalloprotease ZMPSTE24

The integral membrane zinc metalloprotease ZMPSTE24 is important for human health and longevity. ZMPSTE24 performs a key proteolytic step in maturation of prelamin A, the precursor of the nuclear scaffold protein lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that prevent cleavage cause the premature aging disease Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid disorders. ZMPSTE24 has a novel structure, with seven transmembrane spans that form a large water-filled membrane chamber whose catalytic site faces the chamber interior. Prelamin A is the only known mammalian substrate for ZMPSTE24, however, the basis of this specificity remains unclear. To define the sequence requirements for ZMPSTE24 cleavage, we mutagenized the eight residues flanking the prelamin A scissile bond (TRSY{downarrow}LLGN) to all other 19 amino acids, creating a library of 152 variants. We also replaced these eight residues with sequences derived from putative ZMPSTE24 cleavage sites from amphibian, bird, and fish prelamin A. Cleavage of prelamin A variants was assessed using an in vivo yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. We found that residues on the C-terminal side of the cleavage site are most sensitive to changes. Consistent with other zinc metalloproteases, including thermolysin, ZMPSTE24 preferred hydrophobic residues at the P1 position (Leu647), but in addition, showed a similar, albeit muted, pattern at P2. Our findings begin to define a consensus sequence for ZMPSTE24 that helps to clarify how this physiologically important protease functions and may ultimately lead to identifying additional substrates.

cell biology↗

bioRxiv2020

Selective Labeling and Identification of the Tumor Cell Proteome of Pancreatic Cancer In Vivo

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers. Dissecting the tumor cell proteome from that of the non-tumor cells in the PDAC tumor bulk is critical for tumorigenesis studies, biomarker discovery, and development of therapeutics. However, investigating the tumor cell proteome has proven evasive due to the tumors extremely complex cellular composition. To circumvent this technical barrier, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and data-independent acquisition mass spectrometry (DIA-MS) in an orthotopic PDAC model to specifically identify the tumor cell proteome in vivo. Utilizing the tumor cell-specific expression of a mutant tRNA synthetase transgene, this approach provides tumor cells with the exclusive ability to incorporate an azide-bearing methionine analog into newly synthesized proteins. The azide-tagged tumor cell proteome is subsequently enriched and purified via a bioorthogonal reaction and then identified and quantified using DIA-MS. Applying this workflow to the orthotopic PDAC model, we have identified thousands of proteins expressed by the tumor cells. Furthermore, by comparing the tumor cell and tumor bulk proteomes, we showed that the approach can distinctly differentiate proteins produced by tumor-cells from non-tumor cells within the tumor microenvironment. Our study, for the first time, reveals the tumor cell proteome of PDAC under physiological conditions, providing broad applications for tumorigenesis, therapeutics, and biomarker studies in various human cancers.

cancer biology↗

bioRxiv2020

A new paradigm for Prelamin A proteolytic processing by ZMPSTE24: the upstream SY^LL cleavage occurs first and there is no CaaX processing by ZMPSTE24

Human ZMPSTE24, an integral membrane zinc metalloprotease, is required for conversion of prelamin A to mature lamin A, a component of the nuclear lamina and failure of this processing causes premature ageing disorders. ZMPSTE24 has also been implicated in both type 2 diabetes mellitus and in viral-host response mechanisms, but to date its only confirmed substrate is the precursor for lamin A. Prelamin A is thought to undergo four C-terminal post-translational modifications in the following order: farnesylation, SIM tripeptide cleavage, carboxymethylation and upstream "SY^LL" cleavage. Here we present evidence that the sequence of events does not follow the accepted dogma. We assessed cleavage of long human prelamin A sequence peptides by purified human ZMPSTE24 combined with FRET and mass spectrometry to detect products. Surprisingly, we found that the "SY^LL" cleavage occurs before and independent of the C-terminal CSIM modifications. We also found that ZMPSTE24 does not perform the predicted C^SIM tripeptide cleavage, but rather it removes an IM dipeptide. ZMPSTE24 can perform a tripeptide cleavage with a canonical CaaX box (C: cysteine; a: aliphatic; X: any residue), but the C-terminus of prelamin A is not a true CaaX sequence. Regardless of the C-terminal modifications of prelamin A, ZMPSTE24 can perform upstream SY^LL cleavage, thus removing the unwanted farnesylated C-terminus. Therefore, it is failure of SY^LL cleavage, not the C-terminal processing that is the likely cause of progeroid disorders.

biochemistry↗

bioRxiv2020

Klp2 and Ase1 synergize to maintain meiotic spindle stability during metaphase I

The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I, but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule crosslinker Ase1 as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.

cell biology↗

bioRxiv2019

Moxifloxacin efficacy against Mycobacterium abscessus in vivo using zebrafish model

Moxifloxacin (MFX) showed good activity in vitro against Mycobacterium abscessus (M. abscessus) and was suggested as one of the antibiotic regimens for adults with M. abscessus disease. However, some other studies showed that MFX showed less or none activity against M. abscessus. In our study we aim to evaluate MFX activity against M. abscessus using zebrafish (ZF) model in vivo. MIC of each drugs were determined by broth microdilution method. M. abscessus labeled by CM-DiI, were micro-injected into ZF. Survival curves were determined by recording dead ZF every day. After 4 days of incubation ZF were lysed. Colony-forming unit (CFU) were enumerated and results are expressed as mean log10 CFU per ZF. Bacteria dissemination and fluorescence intensity in ZF were observed and analyzed. Inhibition rate was also calculated. In our study MFX showed good activity in vitro. But in vivo MFX showed limited restriction to M. abscessus. The association between increased survival and high dose of MFX is not significant. Same results were observed in bacterial fluorescence intensity and inhibition rates, with no significant difference when compared with no drug group (P > 0.05). However, significant difference was observed in azithromycin (AZM) group. MFX showed limited efficacy on Mycobacterium abscessus in vivo using ZF model. MFXs activity in vivo need to be confirmed.

microbiology↗

bioRxiv2019

C17orf53 defines a novel pathway involved in inter-strand crosslink repair

ATR kinase is a master regulator of genome maintenance and participates in DNA replication and various DNA repair pathways. In a genome-wide screening for ATR-dependent fitness genes, we identified a previously uncharacterized gene, C17orf53, whose loss led to hypersensitivity to ATR inhibition. C17orf53 is conserved in vertebrate and is required for efficient cell proliferation. Loss of C17orf53 slowed down DNA replication and led to pronounced ICL repair defect. Further genetic analyses revealed that C17orf53 functions downstream in ICL repair pathway, probably by affecting the loading of repair factors such as RAD54. In addition, we showed that C17orf53 is a ssDNA- and RPA-binding protein, both of which are important for its functions in the cell. Taken together, C17orf53 is a novel component involved in ICL repair pathway.

biochemistry↗

bioRxiv2017

ZMPSTE24 Missense Mutations that Cause Progeroid Diseases Decrease Prelamin A Cleavage Activity, Protein Stability, or Both

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein critical for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA. After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor, prelamin A, undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) and the related progeroid disorders mandibuloacral dysplasia-type B (MAD-B) and restrictive dermopathy (RD). Here we report a \"humanized yeast\" system to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability, or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in ER-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib, which may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this \"humanized yeast system\" can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.

cell biology↗