Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.

An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation.

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.

Microbial metabolism of L-tyrosine protects against allergic airway inflammation.

The constituents of the gut microbiome are determined by the local habitat, which itself is shaped by immunological pressures, such as mucosal IgA. Using a mouse model of restricted antibody repertoire, we identified a role for antibody-microbe interactions in shaping a community of bacteria with an enhanced capacity to metabolize L-tyrosine. This model led to increased concentrations of p-cresol sulfate (PCS), which protected the host against allergic airway inflammation. PCS selectively reduced CCL20 production by airway epithelial cells due to an uncoupling of epidermal growth factor receptor (EGFR) and Toll-like receptor 4 (TLR4) signaling. Together, these data reveal a gut microbe-derived metabolite pathway that acts distally on the airway epithelium to reduce allergic airway responses, such as those underpinning asthma.

Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination.

Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.

Evidence for Pro-angiogenic Functions of VEGF-Ax.

The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1.

Phase transition of spindle-associated protein regulate spindle apparatus assembly.

Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks.

Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling

AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.

Covalent targeted radioligands potentiate radionuclide therapy.

Targeted radionuclide therapy, in which radiopharmaceuticals deliver potent radionuclides to tumours for localized irradiation, has addressed unmet clinical needs and improved outcomes for patients with cancer1-4. A therapeutic radiopharmaceutical must achieve both sustainable tumour targeting and fast clearance from healthy tissue, which remains a major challenge5,6. A targeted ligation strategy that selectively fixes the radiopharmaceutical to the target protein in the tumour would be an ideal solution. Here we installed a sulfur (VI) fluoride exchange (SuFEx) chemistry-based linker on radiopharmaceuticals to prevent excessively fast tumour clearance. When the engineered radiopharmaceutical binds to the tumour-specific protein, the system undergoes a binding-to-ligation transition and readily conjugates to the tyrosine residues through the 'click' SuFEx reaction. The application of this strategy to a fibroblast activation protein (FAP) inhibitor (FAPI) triggered more than 80% covalent binding to the protein and almost no dissociation for six days. In mice, SuFEx-engineered FAPI showed 257% greater tumour uptake than did the original FAPI, and increased tumour retention by 13-fold. The uptake in healthy tissues was rapidly cleared. In a pilot imaging study, this strategy identified more tumour lesions in patients with cancer than did other methods. SuFEx-engineered FAPI also successfully achieved targeted ß- and α-radionuclide therapy, causing nearly complete tumour regression in mice. Another SuFEx-engineered radioligand that targets prostate-specific membrane antigen (PSMA) also showed enhanced therapeutic efficacy. Considering the broad scope of proteins that can potentially be ligated to SuFEx warheads, it might be possible to adapt this strategy to other cancer targets.

Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Metabolic shift induced by systemic activation of T cells in PD-1-deficient mice perturbs brain monoamines and emotional behavior.

T cells reorganize their metabolic profiles after being activated, but the systemic metabolic effect of sustained activation of the immune system has remained unexplored. Here we report that augmented T cell responses in Pdcd1-/- mice, which lack the inhibitory receptor PD-1, induced a metabolic serum signature characterized by depletion of amino acids. We found that the depletion of amino acids in serum was due to the accumulation of amino acids in activated Pdcd1-/- T cells in the lymph nodes. A systemic decrease in tryptophan and tyrosine led to substantial deficiency in the neurotransmitters serotonin and dopamine in the brain, which resulted in behavioral changes dominated by anxiety-like behavior and exacerbated fear responses. Together these data indicate that excessive activation of T cells causes a systemic metabolomic shift with consequences that extend beyond the immune system.

Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility.

Spermatozoa must leave one organism, navigate long distances, and deliver their paternal DNA into a mature egg. For successful navigation and delivery, a sperm-specific calcium channel is activated in the mammalian flagellum. The genes encoding this channel (CatSpers) appear first in ancient uniflagellates, suggesting that sperm use adaptive strategies developed long ago for single-cell navigation. Here, using genetics, super-resolution fluorescence microscopy, and phosphoproteomics, we investigate the CatSper-dependent mechanisms underlying this flagellar switch. We find that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we find unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility.

Tyrosine - a structural glue for hierarchical protein assembly.

Protein self-assembly, guided by the interplay of sequence- and environment-dependent liquid-liquid phase separation (LLPS), constitutes a fundamental process in the assembly of numerous intrinsically disordered proteins. Heuristic examination of these proteins has underscored the role of tyrosine residues, evident in their conservation and pivotal involvement in initiating LLPS and subsequent liquid-solid phase transitions (LSPT). The development of tyrosine-templated constructs, designed to mimic their natural counterparts, emerges as a promising strategy for creating adaptive, self-assembling systems with diverse applications. This review explores the central role of tyrosine in orchestrating protein self-assembly, delving into key interactions and examining its potential in innovative applications, including responsive biomaterials and bioengineering.

Visualizing protein breathing motions associated with aromatic ring flipping.

Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips-that is, 180° rotations-despite their role in maintaining the protein fold1-3. It was suggested that large-scale 'breathing' motions of the surrounding protein environment would be necessary to accommodate these ring flipping events1. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.

The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation.

Deregulation of the TH17 subset of helper T cells is closely linked with immunological disorders and inflammatory diseases. However, the mechanism by which TH17 cells are regulated remains elusive. Here we found that the phosphatase DUSP2 (PAC1) negatively regulated the development of TH17 cells. DUSP2 was directly associated with the signal transducer and transcription activator STAT3 and attenuated its activity through dephosphorylation of STAT3 at Tyr705 and Ser727. DUSP2-deficient mice exhibited severe susceptibility to experimental colitis, with enhanced differentiation of TH17 cells and secretion of proinflammatory cytokines. In clinical patients with ulcerative colitis, DUSP2 was downregulated by DNA methylation and was not induced during T cell activation. Our data demonstrate that DUSP2 is a true STAT3 phosphatase that modulates the development of TH17 cells in the autoimmune response and inflammation.

The extracellular matrix supports breast cancer cell growth under amino acid starvation by promoting tyrosine catabolism.

Breast tumours are embedded in a collagen I-rich extracellular matrix (ECM) network, where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required for cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required macropinocytosis-dependent ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the up-regulation of the intracellular levels of several amino acids, most notably tyrosine and phenylalanine. This resulted in elevated tyrosine catabolism on ECM under starvation, leading to increased fumarate levels, potentially feeding into the tricarboxylic acid (TCA) cycle. Interestingly, this pathway was required for ECM-dependent cell growth and invasive cell migration under amino acid starvation, as the knockdown of p-hydroxyphenylpyruvate hydroxylase-like protein (HPDL), the third enzyme of the pathway, opposed cell growth and motility on ECM in both 2D and 3D systems, without affecting cell proliferation on plastic. Finally, high HPDL expression correlated with poor prognosis in breast cancer patients. Collectively, our results highlight that the ECM in the tumour microenvironment (TME) represents an alternative source of nutrients to support cancer cell growth by regulating phenylalanine and tyrosine metabolism.

The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway.

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.

SHP2 regulates GluA2 tyrosine phosphorylation required for AMPA receptor endocytosis and mGluR-LTD.

Posttranslational modifications regulate the properties and abundance of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that mediate fast excitatory synaptic transmission and synaptic plasticity in the central nervous system. During long-term depression (LTD), protein tyrosine phosphatases (PTPs) dephosphorylate tyrosine residues in the C-terminal tail of AMPA receptor GluA2 subunit, which is essential for GluA2 endocytosis and group I metabotropic glutamate receptor (mGluR)-dependent LTD. However, as a selective downstream effector of mGluRs, the mGluR-dependent PTP responsible for GluA2 tyrosine dephosphorylation remains elusive at Schaffer collateral (SC)-CA1 synapses. In the present study, we find that mGluR5 stimulation activates Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) by increasing phospho-Y542 levels in SHP2. Under steady-state conditions, SHP2 plays a protective role in stabilizing phospho-Y869 of GluA2 by directly interacting with GluA2 phosphorylated at Y869, without affecting GluA2 phospho-Y876 levels. Upon mGluR5 stimulation, SHP2 dephosphorylates GluA2 at Y869 and Y876, resulting in GluA2 endocytosis and mGluR-LTD. Our results establish SHP2 as a downstream effector of mGluR5 and indicate a dual action of SHP2 in regulating GluA2 tyrosine phosphorylation and function. Given the implications of mGluR5 and SHP2 in synaptic pathophysiology, we propose SHP2 as a promising therapeutic target for neurodevelopmental and autism spectrum disorders.