O‐GlcNAcylation of TDP‐43 suppresses proteinopathies and promotes TDP‐43’s mRNA splicing activity

Pathological TDP‐43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD‐TDP); however, how TDP‐43 aggregation and function are regulated remain poorly understood. Here, we show that O‐GlcNAc transferase OGT‐mediated O‐GlcNAcylation of TDP‐43 suppresses ALS‐associated proteinopathies and promotes TDP‐43''s splicing function. Biochemical and cell‐based assays indicate that OGT''s catalytic activity suppresses TDP‐43 aggregation and hyperphosphorylation, whereas abolishment of TDP‐43 O‐GlcNAcylation impairs its RNA splicing activity. We further show that TDP‐43 mutations in the O‐GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP‐43 overexpression in Drosophila motor neurons. We finally demonstrate that O‐GlcNAcylation of TDP‐43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O‐GlcNAcylation might be a target for the treatment of TDP‐43‐linked pathogenesis.     

FTLD‐TDP assemblies seed neoaggregates with subtype‐specific features via a prion‐like cascade

Morphologically distinct TDP‐43 aggregates occur in clinically different FTLD‐TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP‐43 follows a prion‐like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD‐TDP‐A and FTLD‐TDP‐C aggregates. Upon inoculation of patient‐derived aggregates in cells, FTLD‐TDP‐A seeds amplify in a template‐dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD‐TDP‐C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N‐to‐C directionality and with subtype‐specific timelines. The resulting FTLD‐TDP‐A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD‐TDP‐C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype‐specific therapeutics.     

Frontotemporal dementia‐linked P112H mutation of TDP‐43 induces protein structural change and impairs its RNA binding function

TDP‐43 forms the primary constituents of the cytoplasmic inclusions contributing to various neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Over 60 TDP‐43 mutations have been identified in patients suffering from these two diseases, but most variations are located in the protein''s disordered C‐terminal glycine‐rich region. P112H mutation of TDP‐43 has been uniquely linked to FTD, and is located in the first RNA recognition motif (RRM1). This mutation is thought to be pathogenic, but its impact on TDP‐43 at the protein level remains unclear. Here, we compare the biochemical and biophysical properties of TDP‐43 truncated proteins with or without P112H mutation. We show that P112H‐mutated TDP‐43 proteins exhibit higher thermal stability, impaired RNA‐binding activity, and a reduced tendency to aggregate relative to wild‐type proteins. Near‐UV CD, 2D‐nuclear‐magnetic resonance, and intrinsic fluorescence spectrometry further reveal that the P112H mutation in RRM1 generates local conformational changes surrounding the mutational site that disrupt the stacking interactions of the W113 side chain with nucleic acids. Together, these results support the notion that P112H mutation of TDP‐43 contributes to FTD through functional impairment of RNA metabolism and/or structural changes that curtail protein clearance.     

HDAC6 inhibition restores TDP‐43 pathology and axonal transport defects in human motor neurons with TARDBP mutations

TDP‐43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP‐43, are one of the common causes of familial ALS. In this study, we investigate TDP‐43 protein behavior in induced pluripotent stem cell (iPSC)‐derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP‐43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP‐43, C‐terminal fragments, and phospho‐TDP‐43. By generating iPSC lines with allele‐specific tagging of TDP‐43, we find that mutant TDP‐43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP‐43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP‐43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP‐43 pathology.     

Disease‐linked TDP‐43 hyperphosphorylation suppresses TDP‐43 condensation and aggregation

Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation.     

SARS‐CoV‐2 nucleocapsid protein phase‐separates with RNA and with human hnRNPs

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA‐binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) condenses with RNA via liquid–liquid phase separation (LLPS) and that N protein can be recruited in phase‐separated forms of human RNA‐binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N‐terminal IDR and central‐linker IDR, as well as the folded C‐terminal oligomerization domain, while the folded N‐terminal domain and the C‐terminal IDR are not required. N protein phase separation is induced by addition of non‐specific RNA. In addition, N partitions in vitro into phase‐separated forms of full‐length human hnRNPs (TDP‐43, FUS, hnRNPA2) and their low‐complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS‐CoV‐2 viral genome packing and in host‐protein co‐opting necessary for viral replication and infectivity.     

DEAD‐box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3‐piRISC

The piRNA amplification pathway in Bombyx is operated by Ago3 and Siwi in their piRISC form. The DEAD‐box protein, Vasa, facilitates Ago3‐piRISC production by liberating cleaved RNAs from Siwi‐piRISC in an ATP hydrolysis‐dependent manner. However, the Vasa‐like factor facilitating Siwi‐piRISC production along this pathway remains unknown. Here, we identify DEAD‐box polypeptide 43 (DDX43) as the Vasa‐like protein functioning in Siwi‐piRISC production. DDX43 belongs to the helicase superfamily II along with Vasa, and it contains a similar helicase core. DDX43 also contains a K‐homology (KH) domain, a prevalent RNA‐binding domain, within its N‐terminal region. Biochemical analyses show that the helicase core is responsible for Ago3‐piRISC interaction and ATP hydrolysis, while the KH domain enhances the ATPase activity of the helicase core. This enhancement is independent of the RNA‐binding activity of the KH domain. For maximal DDX43 RNA‐binding activity, both the KH domain and helicase core are required. This study not only provides new insight into the piRNA amplification mechanism but also reveals unique collaborations between the two domains supporting DDX43 function within the pathway.     

LncRNA HCG11/miR‐26b‐5p/QKI5 feedback loop reversed high glucose‐induced proliferation and angiogenesis inhibition of HUVECs

Acute coronary syndrome caused by the rupture of atherosclerotic plaques is one of the primary causes of cerebrovascular and cardiovascular events. Neovascularization within the plaque is closely associated with its stability. Long non‐coding RNA (lncRNA) serves a crucial role in regulating vascular endothelial cells (VECs) proliferation and angiogenesis. In this study, we identified lncRNA HCG11, which is highly expressed in patients with vulnerable plaque compared with stable plaque. Then, functional experiments showed that HCG11 reversed high glucose‐induced vascular endothelial injury through increased cell proliferation and tube formation. Meanwhile, vascular‐related RNA‐binding protein QKI5 was greatly activated. Luciferase reporter assays and RNA‐binding protein immunoprecipitation (RIP) assays verified interaction between them. Interestingly, HCG11 can also positively regulated by QKI5. Bioinformatics analysis and luciferase reporter assays showed HCG11 can worked as a competing endogenous RNA by sponging miR‐26b‐5p, and QKI5 was speculated as the target of miR‐26b‐5p. Taken together, our findings revered that the feedback loop of lncRNA HCG11/miR‐26b‐5p/QKI‐5 played a vital role in the physiological function of HUVECs, and this also provide a potential target for therapeutic strategies of As.     

Opposing activities of IFITM proteins in SARS‐CoV‐2 infection

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.     

A structural inventory of native ribosomal ABCE1‐43S pre‐initiation complexes

In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub‐complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP‐binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre‐initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1‐containing pre‐initiation complexes by cryo‐EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide‐binding domains, while interacting with the N‐terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C‐terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near‐complete molecular picture of the architecture and sophisticated interaction network of the 43S‐bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.     

DNA asymmetry promotes SUMO modification of the single‐stranded DNA‐binding protein RPA

Repair of DNA double‐stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post‐translational modification of repair factors. In budding yeast, single‐stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA–RPA complexes are then SUMO‐modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3'' single‐stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO‐RPA signal is amplified by successive rounds of Siz2‐dependent SUMO modification of RPA and dissociation of SUMO‐RPA at the junction between single‐ and double‐stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.     

Circ_0000052/miR‐382‐3p axis induces PD‐L1 expression and regulates cell proliferation and immune evasion in head and neck squamous cell carcinoma

A better understanding of the mechanisms underlying PD‐L1 aberrant expression in head and neck squamous cell carcinoma (HNSCC) will help reveal predictive biomarkers and overcome resistance to treatment. In this study, the prognostic significance of PD‐L1 in forty‐five HNSCC archival samples was determined by qRT‐PCR. The biological function associated with malignant behaviour was assessed by PD‐L1 depletion, miR‐382‐3p re‐expression and regulation of circ_0000052. The interactions of PD‐L1‐miRNA and miRNA‐circRNA were determined by qRT‐PCR, Western blot analysis, dual‐luciferase reporter assays and RNA immunoprecipitation assays. PD‐L1 was highly expressed in patient samples and cancer cell lines. Higher levels of PD‐L1 were associated with patient recurrences and play a pivotal role in regulating cell proliferation, migration, invasion, clonogenicity and apoptosis. In addition to demonstrating that the IFN‐γ/JAK2/STAT1 signalling pathway can induce PD‐L1 overexpression in HNSCC, a novel mechanism by which upregulated circ_0000052 mediates PD‐L1 overexpression was also demonstrated. To do this, circ_0000052 competitively binds to miR‐382‐3p and alleviates its repression of PD‐L1. This leads to overexpression of PD‐L1, causing the aggressiveness of the cells. Our data demonstrate that circ_0000052 is oncogenic, and the circ_0000052/miR‐382‐3p/PD‐L1 axis is critical in HNSCC progression. The manipulation of circRNAs/miRNAs in combination with anti‐PD‐L1 therapy may improve personalized disease management.     

SARS‐CoV‐2 sensing by RIG‐I and MDA5 links epithelial infection to macrophage inflammation

SARS‐CoV‐2 infection causes broad‐spectrum immunopathological disease, exacerbated by inflammatory co‐morbidities. A better understanding of mechanisms underpinning virus‐associated inflammation is required to develop effective therapeutics. Here, we discover that SARS‐CoV‐2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG‐I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS‐CoV‐2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS‐CoV‐2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation‐associated COVID‐19.     

The CTLA‐4 immune checkpoint protein regulates PD‐L1:PD‐1 interaction via transendocytosis of its ligand CD80

CTLA‐4 and PD‐1 are key immune checkpoint receptors that are targeted in the treatment of cancer. A recently identified physical interaction between the respective ligands, CD80 and PD‐L1, has been shown to block PD‐L1/PD‐1 binding and to prevent PD‐L1 inhibitory functions. Since CTLA‐4 is known to capture and degrade its ligands via transendocytosis, we investigated the interplay between CD80 transendocytosis and CD80/PD‐L1 interaction. We find that transendocytosis of CD80 results in a time‐dependent recovery of PD‐L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is internalised, while its heterodimeric PD‐L1 partner remains on the plasma membrane of the antigen‐presenting cell (APC). CTLA‐4 interactions with CD80 do not appear to be inhibited by PD‐L1, but efficient removal of CD80 requires an intact CTLA‐4 cytoplasmic domain, distinguishing this process from more general trogocytosis and simple CTLA‐4 binding to CD80/PD‐L1 complexes. These data are consistent with CTLA‐4 acting as modulator of PD‐L1:PD‐1 interactions via control of CD80.     

miR‐4286 functions in osteogenesis and angiogenesis via targeting histone deacetylase 3 and alleviates alcohol‐induced bone loss in mice

ObjectivesAlcohol consumption is one of the leading factors contributing to premature osteopenia. MicroRNA (miRNA) coordinates a cascade of anabolic and catabolic processes in bone homeostasis and dynamic vascularization. The aim was to investigate the protective role of miR‐4286 in alcohol‐induced bone loss and its mechanism.Materials and MethodsThe effect of miR‐4286 and alcohol on bone mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) was explored via multiple in vitro assays, including cell proliferation, QPCR, Western blot, osteogenesis, angiogenesis etc miR‐4286 directly regulated HDAC3 was investigated by luciferase reporter assay, and the function of HDAC3 was also explored in vitro. Moreover, alcohol‐induced bone loss in mice was established to reveal the preventive effect of miR‐4286 by radiographical and histopathological assays.ResultsIn vitro, ethanol dramatically inhibited the proliferation and osteogenesis of BMSCs, and substantially impaired the proliferation and vasculogenesis of HUVECs. However, a forced overexpression of miR‐4286 within BMSCs and HUVECs could largely abolish inhibitory effects by alcohol. Furthermore, alcohol‐induced inhibition on osteogenic and vasculogenic functions was mediated by histone deacetylase 3 (HDAC3), and dual‐luciferase reporter assay showed that HDAC3 was the direct binding target of miR‐4286. In vivo, micro‐CT scanning and histology assessment revealed that miR‐4286 could prevent alcohol‐induced bone loss.ConclusionsWe firstly demonstrated that miR‐4286 might function via intimate osteogenesis‐angiogenesis pathway to alleviate alcohol‐induced osteopenia via targeting HDAC3.     

Biochemically validated structural model of the 15‐subunit intraflagellar transport complex IFT‐B

Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT‐A and IFT‐B that are transported by molecular motors. The IFT‐B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high‐resolution structures are only available for smaller IFT‐B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15‐subunit IFT‐B complex. The model was validated using cross‐linking/mass‐spectrometry data on reconstituted IFT‐B complexes, X‐ray scattering in solution, diffraction from crystals as well as site‐directed mutagenesis and protein‐binding assays. The IFT‐B structure reveals an elongated and highly flexible complex consistent with cryo‐electron tomographic reconstructions of IFT trains. The IFT‐B complex organizes into IFT‐B1 and IFT‐B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT‐B1 complex. Our data present a structural framework to understand IFT‐B complex assembly, function, and ciliopathy variants.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

Efficient degradation of by‐products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER‐associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER‐phagy receptors for ER‐to‐lysosome‐associated degradation (ERLAD). Demannosylation of N‐linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro‐collagen that cycles of de‐/re‐glucosylation of selected N‐glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD‐resistant misfolded proteins for FAM134B‐driven lysosomal delivery. In summary, we show that mannose and glucose processing of N‐glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.