Introduction
Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no ...cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging.
Methods
In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy.
Results
Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3).
Discussion
The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.
The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning ...transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.
Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of ...pro-inflammatory cytokines through activating NF-κB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-κB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-κB activation downstream of NOD engagement. Despite only delaying NF-κB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.
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•First complete observation of encystation at the proteomic level up to mature cyst.•Reproducibly identified 3863 (64%) and quantified 3382 proteins (56%) of Giardia annotated ...proteome across encystation.•One-third (1169 proteins) of the quantified proteome is differentially expressed in mature cysts relative to trophozoites.•Transcriptome-proteome correlation of trophozoites and cysts showed evidence of post-transcriptional regulation.•Encystation is mechanistically complex and tightly regulated.
Cyst formation in the parasitic protist Giardia duodenalis is critical to its transmission. Existing proteomic data quantifies only 17% of coding genes transcribed during encystation and does not cover the complete process from trophozoite to mature cyst. Using high-resolution mass spectrometry, we have quantified proteomic changes across encystation and compared this with published transcriptomic data. We reproducibly identified 3863 (64.5% of Giardia proteins) and quantified 3382 proteins (56.5% of Giardia proteins) over standard trophozoite growth (TY), during low-bile encystation priming (LB), 16 h into encystation (EC), and at cyst maturation (C). This work provides the first known expanded observation of encystation at the proteomic level and triples the coverage of previous encystation proteomes. One-third (1169 proteins) of the quantified proteome is differentially expressed in the mature cyst relative to the trophozoite, including proteasomal machinery, metabolic pathways, and secretory proteins. Changes in lipid metabolism indicated a shift in lipid species dependency during encystation. Consistent with this, we identified the first, putative lipid transporters in this species, representing the steroidogenic acute regulatory protein–related lipid transfer (StARkin), oxysterol binding protein related protein (ORP/Osh) and glycosphingolipid transfer protein (GLTP) families, and follow their differential expression over cyst formation. Lastly, we undertook correlation analyses of the transcriptome and proteome of trophozoites and cysts, and found evidence of post-transcriptional regulation of key protein classes (RNA binding proteins) and stage-specific genes (encystation markers) implicating translation-repression in encystation. We provide the most extensive proteomic analysis of encystation in Giardia to date and the first known exploration across its complete duration. This work identifies encystation as highly coordinated, involving major changes in proteostasis, metabolism and membrane dynamics, and indicates a potential role for post-transcriptional regulation, mediated through RNA-binding proteins. Together our work provides a valuable resource for Giardia research and the development of transmission-blocking anti-giardials.
MARCH proteins are membrane‐associated Ring‐CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as ...immune co‐stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin‐6 (IL6Rα). Here we use over‐expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein‐specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4β1 (VLA4 or VCAM‐1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B‐lymphocytes and this was accompanied by decreased numbers of B‐cells in the spleen.
Autophagy, the process of elimination of cellular components by lysosomal degradation, is essential for animal development and homeostasis. Using the autophagy-dependent Drosophila larval midgut ...degradation model we identified an autophagy regulator, the RING domain ubiquitin ligase CG14435 (detour). Depletion of detour resulted in increased early-stage autophagic vesicles, premature tissue contraction, and overexpression of detour or mammalian homologues, ZNRF1 and ZNRF2, increased autophagic vesicle size. The ablation of ZNRF1 or ZNRF2 in mammalian cells increased basal autophagy. We identified detour interacting proteins including HOPS subunits, deep orange (dor/VPS18), Vacuolar protein sorting 16A (VPS16A), and light (lt/VPS41) and found that detour promotes their ubiquitination. The detour mutant accumulated autophagy-related proteins in young adults, displayed premature ageing, impaired motor function, and activation of innate immunity. Collectively, our findings suggest a role for detour in autophagy, likely through regulation of HOPS complex, with implications for healthy aging.
Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a ...number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2
mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1
). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: K
(ATP), K
(substrate), k
, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations.
Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain ...containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT, and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R. K270 was found to be ubiquitinated with K‐29 polyubiquitin chains by the ubiquitin ligase Nedd4‐2. Thus, our results uncover a novel role of K‐29 polyubiquitin chains in Arrdc4‐mediated EV biogenesis and protein trafficking.
Signalling by the βc family of cytokines Hercus, Timothy R; Dhagat, Urmi; Kan, Winnie L.T ...
Cytokine & growth factor reviews,
06/2013, Letnik:
24, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Abstract The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the βc family due to their receptors sharing the signalling subunit βc, regulates multiple biological processes such as native ...and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the βc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the βc receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities.
Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, ...direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α‐arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4‐mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4–/– mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two‐cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4–/‐ epididymal epithelial cells, and further, supplementation of Arrdc4–/– sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis.
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