PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells.

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

Structural and functional analysis of novel human cytochrome C targets in apoptosis.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.

New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants.

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.

Lysosomes: multifunctional compartments ruled by a complex regulatory network.

More than 50 years have passed since Nobel laureate Cristian de Duve described for the first time the presence of tiny subcellular compartments filled with hydrolytic enzymes: the lysosome. For a long time, lysosomes were deemed simple waste bags exerting a plethora of hydrolytic activities involved in the recycling of biopolymers, and lysosomal genes were considered to just be simple housekeeping genes, transcribed in a constitutive fashion. However, lysosomes are emerging as multifunctional signalling hubs involved in multiple aspects of cell biology, both under homeostatic and pathological conditions. Lysosomes are involved in the regulation of cell metabolism through the mTOR/TFEB axis. They are also key players in the regulation and onset of the immune response. Furthermore, it is becoming clear that lysosomal hydrolases can regulate several biological processes outside of the lysosome. They are also implicated in a complex communication network among subcellular compartments that involves intimate organelle-to-organelle contacts. Furthermore, lysosomal dysfunction is nowadays accepted as the causative event behind several human pathologies: low frequency inherited diseases, cancer, or neurodegenerative, metabolic, inflammatory, and autoimmune diseases. Recent advances in our knowledge of the complex biology of lysosomes have established them as promising therapeutic targets for the treatment of different pathologies. Although recent discoveries have started to highlight that lysosomes are controlled by a complex web of regulatory networks, which in some cases seem to be cell- and stimuli-dependent, to harness the full potential of lysosomes as therapeutic targets, we need a deeper understanding of the little-known signalling pathways regulating this subcellular compartment and its functions.

Nucleus-translocated mitochondrial cytochrome c liberates nucleophosmin-sequestered ARF tumor suppressor by changing nucleolar liquid-liquid phase separation.

The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins.

IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.

Identifying cytokine signaling signatures in primary human Th-1 cells by phospho-proteomics analysis.

Stable isotope labeling by amino acid-based high-resolution phosphoproteomics is a powerful technique that allows for direct comparison of cells stimulated under different experimental conditions. This feature makes it the ideal methodology to identify cytokine signaling networks. Here, we present an optimized protocol for the isolation and identification of phosphopeptides from IL-6-stimulated primary human Th-1 cells. For complete details on the use and execution of this protocol, please refer to Martinez-Fabregas et al. (2020).

Competitive binding of STATs to receptor phospho-Tyr motifs accounts for altered cytokine responses.

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci.

Cytokines are highly pleiotropic ligands that regulate the immune response. Here, using interleukin-6 (IL-6) as a model system, we perform detailed phosphoproteomic and transcriptomic studies in human CD4+ T helper 1 (Th-1) cells to address the molecular bases defining cytokine functional pleiotropy. We identify CDK8 as a negative regulator of STAT3 transcriptional activities, which interacts with STAT3 upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecule inhibitors, reduces the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome is increased upon CDK8 inhibition, which results in a concomitant increase in STAT3-mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions results in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity by modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses.

Kinetics of cytokine receptor trafficking determine signaling and functional selectivity.

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

Mapping Determinants of Cytokine Signaling via Protein Engineering.

Cytokines comprise a large family of secreted ligands that are critical for the regulation of immune homeostasis. Cytokines initiate signaling via dimerization or oligomerization of the cognate receptor subunits, triggering the activation of the Janus Kinases (JAKs)/ signal transducer and activator of transcription (STATs) pathway and the induction of specific gene expression programs and bioactivities. Deregulation of cytokines or their downstream signaling pathways are at the root of many human disorders including autoimmunity and cancer. Identifying and understanding the mechanistic principles that govern cytokine signaling will, therefore, be highly important in order to harness the therapeutic potential of cytokines. In this review, we will analyze how biophysical (ligand-receptor binding geometry and affinity) and cellular (receptor trafficking and intracellular abundance of signaling molecules) parameters shape the cytokine signalosome and cytokine functional pleiotropy; from the initial cytokine binding to its receptor to the degradation of the cytokine receptor complex in the proteasome and/or lysosome. We will also discuss how combining advanced protein engineering with detailed signaling and functional studies has opened promising avenues to tackle complex questions in the cytokine signaling field.

Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis.

Diverse cellular processes depend on the lysosomal protease system but how cells regulate lysosomal proteolytic capacity is only partly understood. We show here that cells can respond to protease/substrate imbalance in this compartment by de novo expression of multiple lysosomal hydrolases. This response, exemplified here either by loss of asparagine endopeptidase (AEP) or other lysosomal cysteine proteases, or by increased endocytic substrate load, is not dependent on the transcription factor EB (TFEB) but rather is triggered by STAT3 activation downstream of lysosomal oxidative stress. Similar lysosomal adaptations are seen in mice and cells expressing a constitutively active form of STAT3. Our results reveal how cells can increase lysosomal protease capacity under 'fed' rather than 'starved' conditions that activate the TFEB system. In addition, STAT3 activation due to lysosomal stress likely explains the hyperproliferative kidney disease and splenomegaly observed in AEP-deficient mice.

Proteomic tools for the analysis of transient interactions between metalloproteins.

Metalloproteins play major roles in cell metabolism and signalling pathways. In many cases, they show moonlighting behaviour, acting in different processes, depending on the physiological state of the cell. To understand these multitasking proteins, we need to discover the partners with which they carry out such novel functions. Although many technological and methodological tools have recently been reported for the detection of protein interactions, specific approaches to studying the interactions involving metalloproteins are not yet well developed. The task is even more challenging for metalloproteins, because they often form short-lived complexes that are difficult to detect. In this review, we gather the different proteomic techniques and biointeractomic tools reported in the literature. All of them have shown their applicability to the study of transient and weak protein-protein interactions, and are therefore suitable for metalloprotein interactions.