Vaccine-induced immune thrombotic thrombocytopenia and spike protein

Background. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome of unclear aetiology occurring after DNA-based vaccinations against COVID-19. The aim of this study was to investigate the DNA vaccine-encoded Sars-cov-2 soluble spike protein (SP). as a potential trigger of platelet activation in VITT. Methods. We studied three VITT patients and seven healthy controls (HCs) within 3 weeks from the first dose of ChAdOx1 nCoV-19, and one non vaccinated HC. Serum levels of SP and soluble angiotensin-converting enzyme 2 (sACE2), ACE2 expression in platelets and platelet response to VITT serum stimulation were studied. A thrombus retrieved during mechanical thrombectomy from one VITT patients, was analysed by immunohistochemistry for SP and ACE2. Neutrophil extracellular traps (NETs) markers and coagulation parameters were also measured. Results. We detected serum SP (up to 35 days post-vaccination) and sACE2 in all VITT patients, and respectively in two and three out of 7 vaccinated HCs. Only platelets from one non-vaccinated HC expressed ACE2. VITT sera markedly activated platelets and this activation was inhibited by both anti-SP and anti-FcγRIIA blocking antibodies. The thrombus showed positive immunohistochemical labelling of platelets using an anti-SP antibody with reduced ACE2 expression, compared to a thrombus from a pre-pandemic stroke patient. Markers of endothelial dysfunction, NETs and hypercoagulability state were present in all VITT sera. Conclusions. The present data provides first evidence that DNA vaccine-encoded Sars-cov-2 SP is detectable in VITT sera (several weeks post-vaccination) and in a platelet-rich thrombus, and that may contribute to the initial platelet stimulation in VITT patients.

Circadian Proteins Cry and Rev-erb Deepen Cellular Quiescence by Down-regulating Cyclin D and Cdk4,6

The proper balance and transition between cellular quiescence and proliferation are critical to tissue homeostasis, and their deregulations are commonly found in many human diseases, including cancer and aging. Recent studies showed that the reentry of quiescent cells to the cell cycle is subjected to circadian regulation. However, the underlying mechanisms are largely unknown. Here, we report that two circadian proteins, Cryptochrome (Cry) and Rev-erb, deepen cellular quiescence in rat embryonic fibroblasts, resulting in stronger serum stimulation required for cells to exit quiescence and reenter the cell cycle. This finding was opposite from what we expected from the literature. By modeling a library of possible regulatory topologies linking Cry and Rev-erb to a bistable Rb-E2f gene network switch that controls the quiescence-to-proliferation transition and by experimentally testing model predictions, we found Cry and Rev-erb converge to downregulate Cyclin D/Cdk4,6 activity, leading to an ultrasensitive increase of the serum threshold to activate the Rb-E2f bistable switch. Our findings suggest a mechanistic role of circadian proteins in modulating the depth of cellular quiescence, which may have implications in the varying potentials of tissue repair and regeneration at different times of the day.

Sufu negatively regulates both initiations of centrosome duplication and DNA replication

Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition.

Codependency and mutual exclusivity for gene community detection from sparse single-cell transcriptome data

Single-cell RNA-seq (scRNA-seq) can be used to characterize cellular heterogeneity in thousands of cells. The reconstruction of a gene network based on coexpression patterns is a fundamental task in scRNA-seq analyses, and the mutual exclusivity of gene expression can be critical for understanding such heterogeneity. Here, we propose an approach for detecting communities from a genetic network constructed on the basis of coexpression properties. The community-based comparison of multiple coexpression networks enables the identification of functionally related gene clusters that cannot be fully captured through differential gene expression-based analysis. We also developed a novel metric referred to as the exclusively expressed index (EEI) that identifies mutually exclusive gene pairs from sparse scRNA-seq data. EEI quantifies and ranks the exclusive expression levels of all gene pairs from binary expression patterns while maintaining robustness against a low sequencing depth. We applied our methods to glioblastoma scRNA-seq data and found that gene communities were partially conserved after serum stimulation despite a considerable number of differentially expressed genes. We also demonstrate that the identification of mutually exclusive gene sets with EEI can improve the sensitivity of capturing cellular heterogeneity. Our methods complement existing approaches and provide new biological insights, even for a large, sparse dataset, in the single-cell analysis field.

hC9ORF78 localizes to kinetochores and is required for proper chromosome segregation

C9ORF78 is a poorly characterized protein found in diverse eukaryotes. Previous work indicated overexpression of hC9ORF78 (aka HCA59) in malignant tissues indicating a possible involvement in growth regulatory pathways. Additional studies in fission yeast and humans uncover a potential function in regulating the spliceosome. In studies of GFP-tagged hC9ORF78 we observed a dramatic reduction in protein abundance in cells grown to confluence and/or deprived of serum growth factors. Serum stimulation induced synchronous re-expression of the protein in HeLa cells. This effect was also observed with the endogenous protein. Overexpressing either E2F1 or N-Myc resulted in elevated hC9ORF78 expression potentially explaining the serum-dependent upregulation of the protein. Immunofluorescence analysis indicates that hC9ORF78 localizes to nuclei in interphase but does not appear to concentrate in speckles as would be expected for a splicing protein. Surprisingly, a subpopulation of hC9ORF78 co-localizes with ACA, Mad1 and Hec1 in mitotic cells suggesting that this protein may associate with kinetochores or centromeres. Furthermore, knocking-down hC9ORF78 caused mis-alignment of chromosomes in mitosis. These studies uncover novel mitotic function and subcellular localization of cancer antigen hC9ORF78.SUMMARY STATEMENThC9ORF78 regulates chromosome segregation.

Kindlin-2 promotes rear focal adhesion disassembly and directional persistence in cell migration

Cell migration involves front-rear asymmetric FA dynamics, which facilitates trailing edge detachment and directional persistence. Here we show that kinldin-2 is critical for FA sliding and disassembly in migrating cells. Loss of kindlin-2 markedly reduced FA number and selectively impaired rear FA sliding and disassembly, resulting in defective rear retraction and reduced directional persistence during cell migration. Kindlin-2 deficient cells failed to develop serum-induced actomyosin-dependent tension at FAs. At the molecular level, kindlin-2 directly interacted with myosin light chain kinase (MLCK), which was enhanced in response to serum stimulation. Serum deprivation inhibited rear FA disassembly, which was released in response to serum stimulation. Overexpression of the MLCK-binding kindlin-2 F0F1 fragment (aa 1-167), which inhibits the interaction of endogenous kindlin-2 with MLCK, phenocopied kindlin-2 deficiency-induced migration defects. Inhibition of MLCK, like loss of kindlin-2, also impaired trailing edge detachment, rear FA disassembly and directional persistence. These results suggest a role of kindlin-2 in promoting actomyosin contractility at FAs, leading to increased rear FA sliding and disassembly and directional persistence in cell migration.

Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF

While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5′ leader of POLG messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5′ of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5′ leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

Protein kinase D1 phosphorylates CBX8 to facilitate the disassociation of PRC1 complex from p16 promoter and promotes cell senescence

The Polycomb group (PcG) protein chromobox 8 (CBX8) is the subunit of Polycomb repressive complex 1 (PRC1) and recognizes the trimethylation of histone H3 on Lysine 27 (H3K27me3), and coordinates with PRC2 complex to function as epigenetic gene silencer. CBX8 plays a key role in cell proliferation, stem cell biology, cell senescence, and cancer development. However, our knowledge of CBX8 post-translational modifications remains elusive. Here, we report that protein kinase D1 (PKD1) interacts and phosphorylates CBX8 at Ser256 and Ser311 in an evolutionarily conserved motif. We found that PKD1 activation triggered by serum stimulation, Nocodazole treatment and oncogene Ras-induced cell senescence (Ras OIS) all promotes CBX8 S256/311 phosphorylation. PKD1-mediated CBX8 S256/311 phosphorylation impairs PRC1 complex integrity by reducing the binding of CBX8 to other PRC1 components BMI1 and RING1B, decreases the monoubiquitination of histone H2AK119, and results in CBX8 dissociation from its target INK4a/ARF locus and the de-repression of p16, and thus ultimately facilitates cellular senescence. CBX8 S256/311 phosphorylation also compromises hepatocellular cancer cells proliferation and migration. Collectively, these results suggest that PKD1-mediated CBX8 S256/311 phosphorylation is a key mechanism governing CBX8 function, including cell senescence and cancer cell proliferation.Financial supportThis work was supported by grants from Ministry of Science and Technology of the People’s Republic of China (2018YFC2000102), and from National Natural Science Foundation of China (31871382 and 81571369).

Radixin Relocalization and Nonmuscle α-Actinin Expression Are Features of Remodeling Cardiomyocytes in Adult Patients with Dilated Cardiomyopathy

Background. Pediatric patients show an impressive capacity of cardiac regeneration. In contrast, severely deteriorated adult hearts do usually not recover. Since cardiac remodeling—involving the expression of fetal genes—is regarded as an adaptation to stress, we compared hearts of adult patients suffering from dilated cardiomyopathy (DCM) with remodeling of cultured neonatal (NRC) as well as adult (ARC) rat cardiomyocytes and the developing postnatal myocardium. Methods. NRC and ARC were stimulated with serum and cardiac morphogens derived from DCM hearts. Protein synthesis (PS) as well as protein accumulation (PA) was measured, and cell survival was determined under ischemic conditions. Fetal markers were investigated by Western blot. Biomarkers of remodeling were analyzed in controls, DCM, and 2- to 6-month-old children with tetralogy of Fallot as well as in neonatal and adult rats by immunofluorescence. Results. In NRC, serum and morphogens strongly stimulated PS and PA and the reestablishment of cell-cell contacts (CCC). In ARC, both stimulants increased PS and CCC, but PA was only elevated after serum stimulation. In contrast to serum, morphogen treatment resulted in the expression of fetal genes in ARC as determined by nonmuscle α-actinin-1 and α-actinin-4 expression (NM-actinins) and was associated with increased survival under ischemia. NM-actinins were present in cardiomyocytes of DCM in a cross-striated pattern reminiscent of sarcomeres as well as in extensions of the area of the intercalated disc (ID). NM-actinins are expressed in NRC and in the developing heart. Radixin staining revealed remodeling of the area of the ID in DCM almost identical to stimulated cultured ARC. Conclusions. Remodeling was similar in ARC and in cardiomyocytes of DCM suggesting evolutionary conserved mechanisms of regeneration. Despite activation of fetal genes, the atrophy of ARC indicates differences in their regenerative capacity from NRC. Cardiac-derived factors induced NM-actinin expression and increased survival of ischemic ARC while circulating molecules were less effective. Identification of these cardiac-derived factors and determination of their individual capacity to heal or damage are of particular importance for a biomarker-guided therapy in adult patients.

Human prefoldin modulates co-transcriptional pre-mRNA splicing

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the cotranslational folding of actin and tubulin monomers. Additional functions of prefoldin in the cell nucleus have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin depletion provoked transcriptional alterations across the genome of human cells. Severe pre-mRNA splicing defects were also detected, particularly under serum stimulation conditions. We found a significant impairment of co-transcriptional splicing during transcription elongation, which explains why the expression of long genes with a high number of introns was affected the most. We detected prefoldin binding to the gene body of transcribed genes and found that its absence caused significant decrease in the levels of Ser2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Moreover, lack of prefoldin reduced the association of the splicing factor U2AF65 with chromatin, an association that is known to be dependent on Ser2 phosphorylation. Altogether the reported results indicate that human prefoldin is able to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing efficiency.Significance StatementPrefoldin is a protein complex conserved from archaea to humans. It is known that human prefoldin facilitates folding of cytoskeleton monomers in the cytoplasm, but its role in the cell nucleus is unclear. We found prefoldin bound to chromatin in transcribed genes and detected accumulation of unspliced pre-mRNA precursors when prefoldin was depleted, particularly in serum-activated cells. We also found that lack of prefoldin decreases the levels of a phosphorylated form of RNA polymerase II that is the best-known marker of transcription elongation. This defect, in turn, impairs recruitment of the splicing factor U2AF65, provoking the uncoupling of pre-mRNA splicing from transcription. These results show that prefoldin is, in addition to a cytoplasmic chaperone, a modulator of co-transcriptional pre-mRNA processing.