Synaptic vesicles can undergo several modes of exocytosis, endocytosis, and trafficking within individual synapses, and their fates may be linked to differences in the vesicular protein composition. Here, we mapped the intrasynaptic distribution of the synaptic vesicle proteins SV2B and SV2A in glutamatergic synapses of the hippocampus using three-dimensional electron microscopy. SV2B is almost completely absent from both docked vesicles and a distinct cluster of vesicles found near the active zone. In contrast, SV2A was found in all domains of the synapse and was slightly enriched near the active zone. SV2B and SV2A were found on the membrane in the peri-active zone, suggesting recycling from both clusters of vesicles. SV2B knockout mice displayed an increased seizure induction threshold only in a model employing high-frequency stimulation. Our data show that glutamatergic synapses generate molecularly distinct populations of synaptic vesicles and are able to maintain them at steep spatial gradients. The almost complete absence of SV2B from vesicles at the active zone of wildtype mice may explain why SV2A has been found to be more important for vesicle release.

Legionella pneumophila is a Gram-negative bacterium ubiquitously present in freshwater environments and causes a serious type of pneumonia called Legionnaires' disease. During infections, L. pneumophila releases >300 effector proteins into host cells through an Icm/Dot type IV secretion system to manipulate the host defense system for survival within the hosts. Notably, certain effector proteins mediate posttranslational modifications (PTMs), serving as useful approaches exploited by L. pneumophila to modify host proteins. Some effectors catalyze the addition of host protein PTMs, while others mediate the removal of PTMs of host proteins. In this review, we summarize L. pneumophila effector-mediated PTMs of host proteins, including phosphorylation, ubiquitination, glycosylation, AMPylation, phosphocholination, methylation, ADP-ribosylation, as well as dephosphorylation, deubiquitination, deAMPylation, deADP-ribosylation, dephosphocholination, and delipidation. We describe their molecular mechanisms and biological functions in the regulation of bacterial growth and Legionella-containing vacuole biosynthesis and in the disruption of host immune and defense machinery.

Circular RNAs (circRNAs) are unique single-stranded RNA molecules that are covalently closed and expressed in a tissue- and cell-specific manner. Most circRNAs are formed by back-splicing of pre-mRNA and have a wide range of functions in cells. As they lack a 5'-cap and a 3'-poly(A) tail, they are classified as noncoding RNAs that act as sponges for miRNAs and RNA-binding proteins. However, recent studies have found that some circRNAs can undergo cap-independent translation, making them capable of encoding proteins through alternative translation initiation mechanisms. CircRNAs are more stable than linear mRNAs due to their unique circular structure. With mRNA-based drugs receiving significant attention in the past two years, mRNA's instability and immunogenicity are major obstacles to its widespread use. Since circRNA is more stable than mRNA and less immunogenic and can be translated in a tissue-specific manner, it is a promising modality for RNA therapy. In this review, we will provide an overview of the biological functions and potential applications of circRNA.

To facilitate its survival, replication, and dissemination, the intracellular pathogen Legionella pneumophila relies on its unique Type IVB secretion system (T4SS) to deliver over 330 effectors to hijack host cell pathways in a spatiotemporal manner. The underlying mechanisms of the effectors and their host targets are largely unexplored due to their little sequence identity to known proteins and functional redundancy. The T4SS effector SidN (Lpg1083) is secreted into host cells during the late infection period. However, to the best of our knowledge, the molecular characterization of SidN has not been previously studied. Herein, we identified SidN as a nuclear envelope-localized effector. Its structure adopts a novel fold and the N-terminal domain is crucial for its specific subcellular localization. Furthermore, we found that SidN is transported by the eukaryotic karyopherin Importin-13 into the nucleus, where it attaches to the N-terminal region of Lamin-B2 to interfere with the integrity of the nuclear envelope, causing nuclear membrane disruption and eventually cell death. Our work provides new insights into the structure and function of an L. pneumophila effector protein, and suggests a potential strategy that the pathogen utilizes to promote cell death and then benefit bacterial escape from the host for secondary infection.

Hyperglycaemia-induced oxidative stress may disrupt insulin secretion and β-cell survival in diabetes mellitus by overproducing reactive oxygen species. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) exhibit anti-oxidant properties. However, the mechanisms by which hUC-MSCs protect β-cells from high glucose-induced oxidative stress remain underexplored. In this study, we showed that intravenous injection of hUC-MSCs engrafted into the injured pancreas and promoted pancreatic β-cell function in a mouse model of type 1 diabetes mellitus. In vitro study revealed that hUC-MSCs attenuated high glucose-induced oxidative stress and prevented β-cell impairment via the Nrf2/HO-1 signaling pathway. Nrf2 knockdown partially blocked the anti-oxidative effect of hUC-MSCs, resulting in β-cell decompensation in a high glucose environment. Overall, these findings provide novel insights into how hUC-MSCs protect β-cells from high glucose-induced oxidative stress.

AFP is the most widely used biomarker for the diagnosis of hepatocellular carcinoma. However, a substantial proportion of HCC patients have either normal or marginally increased AFP levels in serum, and the underlying mechanisms are not fully understood. In the present study, we provided in vitro as well as in vivo evidence that heat shock protein gp96 promoted AFP expression at the transcriptional level in HCC. NR5A2 was identified as a key transcription factor regulated by AFP and its stability was enhanced by gp96. A further mechanistic study by CO-IP, GST-pull down and molecular docking showed the competitive binding of gp96 and SUMO E3 ligase RanBP2 to NR5A2 at the sites spanning from aa 507 to 539. The binding of gp96 inhibited SUMOylating, ubiquitination, and subsequent degradation of NR5A2. In addition, clinical analysis of HCC patients indicated that gp96 expression was positively correlated to serum AFP levels in tumors. Therefore, our study uncovered the novel regulatory mechanism of gp96 on the stability of its client proteins by directly affecting their SUMOylation and ubiquitination. These findings will help in designing more accurate AFP-based HCC diagnosis and progression monitoring approaches.

Brain-specific serine/threonine-protein kinase 2 (BRSK2) plays critical roles in insulin secretion and β-cell biology. Whether BRSK2 is associated with human type 2 diabetes mellitus (T2DM) is never appreciated. Here, we report that BRSK2 genetic variants are closely related to worsening glucose metabolism due to hyperinsulinemia and insulin resistance in Chinese population. The BRSK2 protein levels are significantly accumulated in β cells from T2DM patients and high-fat-diet (HFD)-fed mice due to enhanced protein stability. Mice with inducible loss-of-function Brsk2 (βKO) are metabolic normal with high potential of insulin secretion under chow-diet condition. Moreover, βKO mice prevent from HFD-induced hyperinsulinemia, obesity, insulin resistance, and glucose intolerance. Conversely, gain-of-function Brsk2 in mature β cells reversibly triggers hyperglycemia due to β-cell hypersecretion-coupled insulin resistance. Mechanistically, BRSK2 senses lipid signals and induces basal insulin secretion in a kinase-dependent manner. The enhanced basal insulin secretion drives insulin resistance and β-cell exhaustion, and thus the onset of T2DM in mice with HFD feeding or β-cell gain-of-function BRSK2. These findings reveal that BRSK2 links hyperinsulinemia to systematic insulin resistance via interplaying between β cells and insulin-sensitive tissues in human genetic variant population or under nutrient-overload conditions.

Exercise intervention at the early stage of type 2 diabetes mellitus (T2DM) can aid in the maintenance of blood glucose homeostasis and prevent the development of macrovascular and microvascular complications. However, the exercise-regulated pathways that prevent the development of T2DM remain largely unclear. In this study, two forms of exercise intervention, treadmill training and voluntary wheel running, were conducted for high-fat diet (HFD)-induced obese mice. We observed that both forms of exercise intervention alleviated HFD-induced insulin resistance and glucose intolerance. Skeletal muscle is recognized as the primary site for postprandial glucose uptake and for responsive alteration beyond exercise training. Metabolomic profiling of the plasma and skeletal muscle in chow, HFD, and HFD-exercise groups revealed robust alterations in metabolic pathways by exercise intervention in both cases. Overlapping analysis identified 9 metabolites, including beta-alanine, leucine, valine, and tryptophan, which were reversed by exercise treatment in both the plasma and skeletal muscle. Transcriptomic analysis of gene expression profiles in the skeletal muscle revealed several key pathways involved in the beneficial effects of exercise on metabolic homeostasis. In addition, integrative transcriptomic and metabolomic analyses uncovered strong correlations between the concentrations of bioactive metabolites and the expression levels of genes involved in energy metabolism, insulin sensitivity, and immune response in the skeletal muscle. This work established two models of exercise intervention in obese mice and provided mechanistic insights into the beneficial effects of exercise intervention on systemic energy homeostasis.

The tumor-suppressive activity of p53 is largely attributed to its ability to induce cell death, including apoptosis, through transcription-dependent and transcription-independent mechanisms. On the one hand, nuclear p53 transcriptionally activates the expression of a myriad of pro-apoptotic BCL-2 family genes, such as NOXA, PUMA, BID, BAD, BIK, BAX, etc., whereas it inactivates the expression of anti-apoptotic BCL-2, BCL-XL, and MCL1, leading to mitochondrial apoptosis. On the other hand, cytoplasmic p53 also promotes mitochondrial apoptosis by directly associating with multiple BCL-2 family proteins in the mitochondria. Apoptosis-related protein in TGF-β signaling pathway (ARTS), a mitochondria-localized pro-apoptotic protein encoded by an alternative spliced variant of the SEPT4 gene, triggers apoptosis by facilitating proteasomal degradation of BCL-2 and XIAP upon pro-apoptotic stimuli. We recently identified SEPT4/ARTS as a new p53 target gene in response to genotoxic stress. ARTS in turn binds to p53, drives its mitochondrial localization, and enhances the interaction between p53 and BCL-XL, thereby promoting mitochondrial apoptosis. This review will illustrate the mechanisms of p53-induced mitochondrial apoptosis, offer some recently discovered new insights into the functions of ARTS in regulating mitochondrial cell death, and discuss the clinical significance of ARTS in cancer and non-cancer diseases.

Understanding how certain animals are capable of regenerating their hearts will provide much needed insights into how this process can be induced in humans in order to reverse the damage caused by myocardial infarction. Currently, it is becoming increasingly evident that cardiac interstitial cells play crucial roles during cardiac regeneration. To understand how interstitial cells behave during this process, we performed single-cell RNA sequencing of regenerating zebrafish hearts. Using a combination of immunohistochemistry, chemical inhibition, and novel transgenic animals, we were able to investigate the role of cell type-specific mechanisms during cardiac regeneration. This approach allowed us to identify a number of important regenerative processes within the interstitial cell populations. Here, we provide detailed insight into how interstitial cells behave during cardiac regeneration, which will serve to increase our understanding of how this process could eventually be induced in humans.

Progressive aggregation of tau protein in neurons is associated with neurodegeneration in tauopathies. Cell non-autonomous disease mechanisms in astrocytes may be important drivers of the disease process but remain largely elusive. Here, we studied cell type-specific responses to intraneuronal tau aggregation prior to neurodegeneration. To this end, we developed a fully human co-culture model of seed-independent intraneuronal tau pathology, which shows no neuron and synapse loss. Using high-content microscopy, we show that intraneuronal tau aggregation induces oxidative stress accompanied by activation of the integrated stress response specifically in astrocytes. This requires the direct co-culture with neurons and is not related to neurodegeneration or extracellular tau levels. Tau-directed antisense therapy reduced intraneuronal tau levels and aggregation and prevented the cell non-autonomous responses in astrocytes. These data identify the astrocytic integrated stress response as a novel disease mechanism activated by intraneuronal tau aggregation. In addition, our data provide the first evidence for the efficacy of tau-directed antisense therapy to target cell autonomous and cell non-autonomous disease pathways in a fully human model of tau pathology.

Lipids and glucose exert many essential physiological functions, such as providing raw materials or energy for cellular biosynthesis, regulating cell signal transduction, and maintaining a constant body temperature. Dysregulation of lipid and glucose metabolism can lead to glucolipid metabolic disorders linked to various metabolic diseases, such as obesity, diabetes, and cardiovascular disease. Therefore, intervention in glucolipid metabolism is a key therapeutic strategy for the treatment of metabolic diseases. Activating transcription factor 3 (ATF3) is a transcription factor that acts as a hub of the cellular adaptive-response network and plays a pivotal role in the regulation of inflammation, apoptosis, DNA repair, and oncogenesis. Emerging evidence has illustrated the vital roles of ATF3 in glucolipid metabolism. ATF3 inhibits intestinal lipid absorption, enhances hepatic triglyceride hydrolysis and fatty acid oxidation, promotes macrophage reverse cholesterol transport, and attenuates the progression of western diet-induced nonalcoholic fatty liver disease and atherosclerosis. In addition to its role in lipid metabolism, ATF3 has also been identified as an important regulator of glucose metabolism. Here, we summarize the recent advances in the understanding of ATF3, mainly focusing on its role in glucose and lipid metabolism and potential therapeutic implications.

The SARS-CoV-2 spike protein contains a functionally important fatty acid (FA) binding site, also found in some other coronaviruses (e.g. SARS-CoV and MERS-CoV), which binds linoleic acid. When occupied by linoleic acid, it reduces infectivity, by 'locking' the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the response of spike variants to linoleic acid removal. D-NEMD simulations show that the FA site is coupled to other, some distant, functional regions of the protein, e.g. the receptor-binding motif, N-terminal domain, furin cleavage site, and regions surrounding the fusion peptide. D-NEMD simulations also identify the allosteric networks connecting the FA site to the functional regions. Comparison of the response of the wild-type spike with four variants (Alpha, Delta, Delta plus, and Omicron BA.1) shows that the variants differ significantly in their response to linoleic acid removal. The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein, with the exception of the receptor-binding motif and the S71-R78 region, which show a weaker link to the FA site. In contrast, Omicron is the most affected variant exhibiting significant differences in the receptor-binding motif, N-terminal domain, V622-L629, and the furin cleavage site. These differences in allosteric modulation may be of functional relevance, potentially affecting transmissibility and virulence. Experimental comparison of the effects of linoleic acid on SARS-CoV-2 variants, including emerging variants, is warranted.

Cancer cell metabolism reprogramming is one of the hallmarks of cancer. Cancer cells preferentially utilize aerobic glycolysis, which is regulated by activated oncogenes and the tumor microenvironment. Extracellular matrix (ECM) in the tumor microenvironment, including the basement membranes (BMs), is dynamically remodeled. However, whether and how ECM regulates tumor glycolysis is largely unknown. We show that type IV collagens, components of BMs essential for the tissue integrity and proper function, are differentially expressed in breast cancer subtypes that α5 chain (α5(IV)) is preferentially expressed in the luminal-type breast cancer and is regulated by estrogen receptor-α. α5(IV) is indispensable for luminal breast cancer development. Ablation of α5(IV) significantly reduces the growth of luminal-type breast cancer cells and impedes the development of luminal-type breast cancer. Impaired cell growth and tumor development capability of α5(IV)-ablated luminal breast cancer cells is attributed to the reduced expression of glucose transporter and glycolytic enzymes and impaired glycolysis in luminal breast cancer cells. Non-integrin collagen receptor discoidin domain receptor-1 (DDR1) expression and p38 mitogen-activated protein kinase activation are attenuated in α5(IV)-ablated luminal breast cancer cells, resulting in reduced c-Myc oncogene expression and phosphorylation. Ectopic expression of constitutively active DDR1 or c-Myc restores the expression of glucose transporter and glycolytic enzymes, and thereafter restores aerobic glycolysis, cell proliferation, and tumor growth of luminal breast cancer. Thus, type IV collagen α5 chain is a luminal-type breast cancer-specific microenvironmental regulator modulating cancer cell metabolism.

YPEL5 is a member of the YPEL gene family that is evolutionarily conserved in the eukaryotic species. To date, the physiological function of YPEL5 has not been yet assessed due to a paucity of genetic animal models. Here, using CRISPR/Cas9-mediated genome editing, we generated a stable ypel5-/- mutant zebrafish line. Disruption of ypel5 expression leads to liver enlargement associated with hepatic cell proliferation. Meanwhile, hepatic metabolism and function are also dysregulated in ypel5-/- mutant as revealed by metabolomic and transcriptomic analysis. Mechanistically, Hnf4a is identified as a crucial downstream mediator and positively regulated by Ypel5. Hnf4a overexpression could largely rescue ypel5 deficiency-induced hepatic defects. Further, PPARα signaling mediates the regulation of Hnf4a by Ypel5 through directly binding to the transcriptional enhancer of Hnf4a gene. Herein, this work demonstrates an essential role for Ypel5 in hepatocyte proliferation and function, and provides the first in vivo evidence for a physiological role of the ypel5 gene in vertebrate.

Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration (nAMD). Myofibroblasts originated from retinal pigment epithelial (RPE) cells through epithelial-mesenchymal transition (EMT) contribute to the fibrosis formation. N6-methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A modification in EMT-related subretinal fibrosis has not yet been elucidated. In this study, we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization, METTL3 was upregulated in RPE cells. Through m6A epitranscriptomic microarray and further verification, high mobility group AT-hook 2 (HMGA2) was identified as the key downstream target of METTL3, subsequently activating potent EMT-inducing transcription factor SNAIL. Finally, by subretinal injections of adeno-associated virus vectors, we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo. In conclusion, our present research identified an epigenetic mechanism of METTL3-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Mono-ADP-ribosylation is a post-translational modification that regulates a variety of biological processes, including DNA damage repair, cell proliferation, metabolism, and stress and immune responses. In mammals, mono-ADP-ribosylation is mainly catalyzed by ADP-ribosyltransferases (ARTs), which consist of two groups: ART cholera toxin like (ARTCs) and ART diphtheria toxin like (ARTDs, also known as PARPs). The human ARTC (hARTC) family is composed of four members: two active mono-ADP-ARTs (hARTC1 and hARTC5) and two enzymatically inactive enzymes (hARTC3 and hARTC4). In this study, we systematically examined the homology, expression, and localization pattern of the hARTC family, with a particular focus on hARTC1. Our results showed that hARTC3 interacted with hARTC1 and promoted the enzymatic activity of hARTC1 by stabilizing hARTC1. We also identified vesicle-associated membrane protein-associated protein B (VAPB) as a new target of hARTC1 and pinpointed Arg50 of VAPB as the ADP-ribosylation site. Furthermore, we demonstrated that knockdown of hARTC1 impaired intracellular calcium homeostasis, highlighting the functional importance of hARTC1-mediated VAPB Arg50 ADP-ribosylation in regulating calcium homeostasis. In summary, our study identified a new target of hARTC1 in the endoplasmic reticulum and suggested that ARTC1 plays a role in regulating calcium signaling.

In mitosis, accurate chromosome segregation depends on super-molecular machinery kinetochore that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN), during mitosis remains uncharacterized. Building on our recent cryo-electron microscopy structure of human CCAN, here we reveal the molecular basis of how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1 kinase, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.

The organized three-dimensional chromosome architecture in the cell nucleus provides the scaffolding for the precise regulation of gene expression. When the cell changes its identity in the cell fate decision-making process, extensive rearrangements of chromosome structures occur accompanied by large-scale adaptations of gene expression, underscoring the importance of chromosome dynamics in shaping genome function. Over the last two decades, the rapid development of experimental methods has provided unprecedented data on characterizing the hierarchical structures and the dynamic properties of chromosomes. In parallel, these enormous data offer valuable opportunities for developing quantitative computational models. Here, we review a variety of large-scale polymer models developed to investigate the structures and dynamics of chromosomes. Different from the underlying modeling strategies, these approaches can be classified into data-driven ("top-down") and physics-based ("bottom-up") categories. We discuss their contributions in offering valuable insights into the relationships among the structures, dynamics, and functions of chromosomes. We highlight the perspectives on future efforts in developing data integration approaches from different experimental technologies and multidisciplinary theoretical/simulation methods combined with different modeling strategies.

Chemoresistance is a primary cause of treatment failure in pancreatic cancer. Identifying cell surface markers specifically expressed in chemoresistant cancer cells (CCCs) could facilitate targeted therapies to overcome chemoresistance. We performed an antibody-based screen and found that TRA-1-60 and TRA-1-81, two 'stemness' cell surface markers, are highly enriched in CCCs. Furthermore, TRA-1-60+/TRA-1-81+ cells are chemoresistant compared to TRA-1-60-/TRA-1-81- cells. Transcriptome profiling identified UGT1A10, shown to be both necessary and sufficient to maintain TRA-1-60/TRA-1-81 expression and chemoresistance. From a high-content chemical screen, we identified Cymarin, which downregulates UGT1A10, eliminates TRA-1-60/TRA-1-81 expression, and increases chemosensitivity both in vitro and in vivo. Finally, TRA-1-60/TRA-1-81 expression is highly specific in primary cancer tissue and positively correlated with chemoresistance and short survival, which highlights their potentiality for targeted therapy. Therefore, we discovered a novel CCC surface marker regulated by a pathway that promotes chemoresistance, as well as a leading drug candidate to target this pathway.