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GRASP65 is a Golgi-associated peripheral protein encoded by the GORASP1 gene and required for Golgi cisternal stacking in vitro. A key role of GRASP65 in the regulation of cell division has also been suggested. However, depletion of GRASP65 in mice has little effect on the Golgi structure and the gene has not been associated with any human phenotype to date. Here, we report the identification of the first human pathogenic variant of GORASP1 (c.1170_1171del; p.Asp390Glufs*18) in a patient combining a neurodevelopmental disorder with neurosensory, neuromuscular, and skeletal abnormalities. Functional analysis revealed that the variant leads to a total absence of GRASP65. The structure of the Golgi apparatus did not show fragmentation, but glycosylation anomalies such as hyposialylation were detected. Mitosis analyses revealed an excess of prometaphases and metaphases with polar chromosomes, suggesting a delay in the cell cycle. These phenotypes were recapitulated in RPE cells in which a similar mutation was introduced by CRISPR/Cas9. These results indicate that loss of GRASP65 in humans causes a novel Golgipathy associated with defects in glycosylation and mitotic progression.

In vivo phage display is a method used for identification of organ- or disease-specific vascular homing peptides for targeted delivery of pharmaceutics. It is agnostic as to the nature and identity of the target molecules. The current in vivo biopanning lacks inbuilt mechanisms to select for peptides capable of vascular homing that would also be capable of tissue penetration to reach therapeutically relevant cells in the tissue parenchyma. Here, we combined in vivo phage display with microdialysis-based parenchymal recovery and high-throughput sequencing to select for peptides that, besides vascular homing, facilitate extravasation and tissue penetration. We first demonstrated in skin wounds that the method can selectively separate known homing peptides from those with additional tissue-penetrating ability. Screening of a naïve peptide library identifies peptides that home and extravasate to extravascular granulation tissue in vascularized and diabetic wounds and cross blood-retina barrier in retinopathy. Our work suggests that in vivo phage display combined with microdialysis can be used for the discovery of vascular homing peptides capable of extravasation and tissue penetration.

IL-17A plays an important role in the pathology of psoriasis and psoriatic arthritis (PsA). However, the pathogenic association between the skin and joint manifestations in PsA is not completely understood. In this study, we initially observed that IL-17A and FGF7 induced endochondral ossification in the mouse entheseal histoculture. Importantly, the responses of endochondral ossification by IL-17A stimulation were strongly inhibited by the treatment of a blocking antibody to FGF receptor 2IIIb, which is the receptor of FGF7, suggesting that FGF7 acts as a downstream factor of IL-17A in the endochondral ossification in the culture. Next, using the animal PsA model, the administration of an anti-FGF receptor 2IIIb antibody resulted in significant suppression of ankylosing enthesitis but not dermatitis. Collectively, our findings indicate that augmented IL-17A in PsA dermatitis induces the elevation of FGF7 levels in joint enthesis and results in a non-redundant role of FGF7 signaling in the development of ankylosing enthesitis in PsA.

Either inhibiting or stabilizing SUMOylation in mitosis causes defects in chromosome segregation, suggesting that dynamic mitotic SUMOylation of proteins is critical to maintain integrity of the genome. Polo-like kinase 1-interacting checkpoint helicase (PICH), a mitotic chromatin remodeling enzyme, interacts with SUMOylated chromosomal proteins via three SUMO-interacting motifs (SIMs) to control their association with chromosomes. Using cell lines with conditional PICH depletion/PICH replacement, we revealed mitotic defects associated with compromised PICH functions toward SUMOylated chromosomal proteins. Defects in either remodeling activity or SIMs of PICH delayed mitotic progression caused by activation of the spindle assembly checkpoint (SAC) indicated by extended duration of Mad1 foci at centromeres. Proteomics analysis of chromosomal SUMOylated proteins whose abundance is controlled by PICH activity identified candidate proteins to explain the SAC activation phenotype. Among the identified candidates, Bub1 kinetochore abundance is increased upon loss of PICH. Our results demonstrated a novel relationship between PICH and the SAC, where PICH directly or indirectly affects Bub1 association at the kinetochore and impacts SAC activity to control mitosis.

The transition of an embryo from gastrulation to organogenesis requires precisely coordinated changes in gene expression, but the underlying mechanisms remain unclear. The RNA-binding protein Trim71 is essential for development and serves as a potent regulator of post-transcriptional gene expression. Here, we show that global deficiency of Trim71 induces severe defects in mesoderm-derived cells at the onset of organogenesis. Murine Trim71-KO embryos displayed impaired primitive erythropoiesis, yolk sac vasculature, heart function, and circulation, explaining the embryonic lethality of these mice. Tie2 ^Cre Trim71 conditional knockout did not induce strong defects, showing that Trim71 expression in endothelial cells and their immediate progenitors is dispensable for embryonic survival. scRNA-seq of E7.5 global Trim71-KO embryos revealed that transcriptomic changes arise already at gastrulation, showing a strong up-regulation of the mesodermal pioneer transcription factor Eomes. We identify Eomes as a direct target of Trim71-mediated mRNA repression via the NHL domain, demonstrating a functional link between these important regulatory genes. Taken together, our data suggest that Trim71-dependent control of gene expression at gastrulation establishes a framework for proper development during organogenesis.

Salamanders demonstrate exceptional resistance to starvation, allowing them to endure extended periods without food in their natural habitats. Although autophagy, a process involving evolutionarily conserved proteins, promotes survival during food scarcity, the specific mechanism by which it contributes to the extreme starvation resistance in newt cells remains unexplored. Our study, using the newt species Pleurodeles waltl, reveals that newt primary fibroblasts maintain constant autophagy activation during prolonged cellular starvation. Unlike normal mammalian fibroblasts, where autophagosome formation increases during acute starvation but returns to baseline levels after extended periods, newt cells maintain elevated autophagosome numbers even 4 d after autophagy initiation, surpassing levels observed in nutrient-rich conditions. Unique P. waltl mTOR orthologs show reduced lysosomal localization compared with mammalian cells in both nutrient-rich and starved states. However, newt cells exhibit dephosphorylation of mTOR substrates under starvation conditions, similar to mammalian cells. These observations suggest that newts may have evolved a distinctive system to balance seemingly conflicting factors: high regenerative capacity and autophagy-mediated survival during starvation.

G-protein-coupled receptors (GPCRs) are virtually involved in every physiological process. However, mechanisms for their ability to regulate a vast array of different processes remain elusive. An unconventional functional modality could at least in part account for such diverse involvements but has yet to be explored. We found HCAR1, a multifunctional lactate GPCR, to localize at the nucleus and therein capable of initiating location-biased signaling notably nuclear-ERK and AKT. We discovered that nuclear HCAR1 (N-HCAR1) is directly involved in regulating diverse processes. Specifically, N-HCAR1 binds to protein complexes that are involved in promoting protein translation, ribosomal biogenesis, and DNA-damage repair. N-HCAR1 also interacts with chromatin remodelers to directly regulate gene expression. We show that N-HCAR1 displays a broader transcriptomic signature than its plasma membrane counterpart. Interestingly, exclusion of HCAR1 from the nucleus has the same effect as its complete cellular depletion on tumor growth and metastasis in vivo. These results reveal noncanonical functions for a cell nucleus-localized GPCR that are distinct from traditional receptor modalities and through which HCAR1 can participate in regulating various cellular processes.

Protein arginine methyltransferase 6 (PRMT6) is a well-characterized epigenetic regulator that methylates histone H3 at arginine 2 (H3R2me2a) in both promoter and enhancer regions, thereby modulating transcriptional initiation. We report here that PRMT6 also regulates gene expression at the post-transcriptional level in the neural pluripotent state and during neuronal differentiation of NT2/D1 cells. PRMT6 knockout causes widespread alternative splicing changes in NT2/D1 cells, most frequently cassette exon alterations. Most of the PRMT6-dependent splicing targets are not transcriptionally affected by the enzyme and regulated in an H3R2me2a-independent manner. However, for a small subset of splicing events, the PRMT6-mediated deposition of H3R2me2a overlaps with the splice site, suggesting a potential dual function in both transcriptional and co-/post-transcriptional regulation. The splicing targets of PRMT6 include ribosomal proteins, splicing factors, and chromatin-modifying enzymes such as PRMT4, DNMT3B, and ASH2L, some of which are associated with differentiation decisions. Taken together, our results in NT2/D1 cells show that PRMT6 exerts predominantly H3R2me2a-independent functions in RNA splicing, which may contribute to pluripotency and neuronal identity.

SARS-CoV-2 can infect cells through endocytic uptake, a process that is targeted by inhibition of lysosomal proteases. However, clinically this approach to treat viral infections has afforded mixed results, with some studies detailing an oral regimen of hydroxychloroquine accompanied by significant off-target toxicities. We rationalized that an organelle-targeted approach will avoid toxicity while increasing the concentration of the drug at the target. Here, we describe a lysosome-targeted, mefloquine-loaded poly(glycerol monostearate-co-ε-caprolactone) nanoparticle (MFQ-NP) for pulmonary delivery via inhalation. Mefloquine is a more effective inhibitor of viral endocytosis than hydroxychloroquine in cellular models of COVID-19. MFQ-NPs are less toxic than molecular mefloquine, are 100-150 nm in diameter, and possess a negative surface charge, which facilitates uptake via endocytosis allowing inhibition of lysosomal proteases. MFQ-NPs inhibit coronavirus infection in mouse MHV-A59 and human OC43 coronavirus model systems and inhibit SARS-CoV-2 WA1 and its Omicron variant in a human lung epithelium model. Organelle-targeted delivery is an effective means to inhibit viral infection.

The plasma membrane covering the primary cilium has a diverse accumulation of receptors and channels. To ensure the sensor function of the cilia, the ciliary membrane has higher cholesterol content than other cell membrane regions. A peroxisomal biogenesis disorder, Zellweger syndrome, characterized by polycystic kidney, is associated with a reduced level of ciliary cholesterol in cells. However, the etiological mechanism by which ciliary cholesterol lowering causes polycystic kidney disease remains unclear. Here, we demonstrated that lowering ciliary cholesterol by either pharmacological treatment or genetic depletion of peroxisomes impairs the localization of a ciliary ion channel polycystin-2. We also generated cultured renal medullary cells and mice carrying a missense variant in the cholesterol-binding site of polycystin-2 detected in the patient database of autosomal dominant polycystic kidney disease. This missense protein showed normal channel activity but decreased localization to the ciliary membrane. The homozygous mice exhibited embryonic lethality and the ciliopathy spectrum conditions of situs inversus and polycystic kidney. Our results suggest that cholesterol controls the ciliary localization of polycystin-2 to prevent polycystic kidney disease.