The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3.

The intracellular bacterial pathogen Coxiella burnetii evades the host response by secreting effector proteins that aid in establishing a replication-friendly niche. Bacterial filamentation induced by cyclic AMP (Fic) enzymes can act as effectors by covalently modifying target proteins with the posttranslational AMPylation by transferring adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl-containing side chain. Here we identify the gene product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at serine 10 and serine 28. We show that CbFic2 acts as a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via a C-terminal helix-turn-helix domain. We propose that CbFic2 performs AMPylation in its monomeric state, switching to a deAMPylating dimer upon DNA binding. This study unveils reversible histone modification by a specific enzyme of a pathogenic bacterium.

Assembly-dependent Structure Formation Shapes Human Interleukin-23 versus Interleukin-12 Secretion.

Interleukin 12 (IL-12) family cytokines connect the innate and adaptive branches of the immune system and regulate immune responses. A unique characteristic of this family is that each member is anα:ßheterodimer. For human αsubunits it has been shown that they depend on theirßsubunit for structure formation and secretion from cells. Since subunits are shared within the family and IL-12 as well as IL-23 use the same ßsubunit, subunit competition may influence cytokine secretion and thus downstream immunological functions. Here, we rationally design a folding-competent human IL-23α subunit that does not depend on itsßsubunit for structure formation. This engineered variant still forms a functional heterodimeric cytokine but shows less chaperone dependency and stronger affinity in assembly with its ßsubunit. It forms IL-23 more efficiently than its natural counterpart, skewing the balance of IL-12 and IL-23 towards more IL-23 formation. Together, our study shows that folding-competent human IL-12 familyαsubunits are obtainable by only few mutations and compatible with assembly and function of the cytokine. These findings might suggest that human α subunits have evolved for assembly-dependent folding to maintain and regulate correct IL-12 family member ratios in the light of subunit competition.

Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions.

Interleukins are secreted proteins that regulate immune responses. Among these, the interleukin 12 (IL-12) family holds a central position in inflammatory and infectious diseases. Each family member consists of an α and a ß subunit that together form a composite cytokine. Within the IL-12 family, IL-35 remains particularly ill-characterized on a molecular level despite its key role in autoimmune diseases and cancer. Here we show that both IL-35 subunits, IL-12α and EBI3, mutually promote their secretion from cells but are not necessarily secreted as a heterodimer. Our data demonstrate that IL-12α and EBI3 are stable proteins in isolation that act as anti-inflammatory molecules. Both reduce secretion of proinflammatory cytokines and induce the development of regulatory T cells. Together, our study reveals IL-12α and EBI3, the subunits of IL-35, to be functionally active anti-inflammatory immune molecules on their own. This extends our understanding of the human cytokine repertoire as a basis for immunotherapeutic approaches.

A chemical probe unravels the reactive proteome of health-associated catechols.

Catechol-containing natural products are common constituents of foods, drinks, and drugs. Natural products carrying this motif are often associated with beneficial biological effects such as anticancer activity and neuroprotection. However, the molecular mode of action behind these properties is poorly understood. Here, we apply a mass spectrometry-based competitive chemical proteomics approach to elucidate the target scope of catechol-containing bioactive molecules from diverse foods and drugs. Inspired by the protein reactivity of catecholamine neurotransmitters, we designed and synthesised a broadly reactive minimalist catechol chemical probe based on dopamine. Initial labelling experiments in live human cells demonstrated broad protein binding by the probe, which was largely outcompeted by its parent compound dopamine. Next, we investigated the competition profile of a selection of biologically relevant catechol-containing substances. With this approach, we characterised the protein reactivity and the target scope of dopamine and ten biologically relevant catechols. Strikingly, proteins associated with the endoplasmic reticulum (ER) were among the main targets. ER stress assays in the presence of reactive catechols revealed an activation of the unfolded protein response (UPR). The UPR is highly relevant in oncology and cellular resilience, which may provide an explanation of the health-promoting effects attributed to many catechol-containing natural products.

Modeling of the human interleukin 12:receptor complex allows to engineer attenuated cytokine variants.

Interleukin 12 (IL-12) plays major roles in immune defense against intracellular pathogens. By activating T cells and increasing antigen presentation, it is also a very potent anti-tumor molecule. Strong immune activation and systemic toxicity, however, so far limit its potential therapeutic use. Building on recent experimental structures of IL-12 related cytokine:receptor complexes, we here provide a high-resolution computational model of the human IL-12:receptor complex. We design attenuated IL-12 variants with lower receptor binding affinities based on molecular dynamics simulations, and subsequently validate them experimentally. These variants show reduced activation of natural killer cells while maintaining T cell activation. This immunological signature is important to develop IL-12 for cancer treatment, where natural killer cells contribute to severe side-effects. Taken together, our study provides detailed insights into structure and dynamics of the human IL-12:receptor complex and leverages them for engineering attenuated variants to elicit fewer side-effects while maintaining relevant biological activity.

Mechanistic insights into the aggregation pathway of the patient-derived immunoglobulin light chain variable domain protein FOR005.

Systemic antibody light chain (AL) amyloidosis is characterized by deposition of amyloid fibrils. Prior to fibril formation, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes. We focus on the patient derived λ-III AL variable domain FOR005 which is mutated at five positions with respect to the closest germline protein. Using solution-state NMR spectroscopy, we follow the individual steps involved in protein misfolding from the native to the amyloid fibril state. Unfavorable mutations in the complementary determining regions introduce a strain in the native protein structure which yields partial unfolding. Driven by electrostatic interactions, the protein converts into a high molecular weight, oligomeric, molten globule. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into fibrils. The topology is determined by balanced electrostatic interactions in the fibril core implying a 180° rotational switch of the beta-sheets around the conserved disulfide bond.

The human signal peptidase complex acts as a quality control enzyme for membrane proteins.

Cells need to detect and degrade faulty membrane proteins to maintain homeostasis. In this study, we identify a previously unknown function of the human signal peptidase complex (SPC)-the enzyme that removes endoplasmic reticulum (ER) signal peptides-as a membrane protein quality control factor. We show that the SPC cleaves membrane proteins that fail to correctly fold or assemble into their native complexes at otherwise hidden cleavage sites, which our study reveals to be abundant in the human membrane proteome. This posttranslocational cleavage synergizes with ER-associated degradation to sustain membrane protein homeostasis and contributes to cellular fitness. Cryptic SPC cleavage sites thus serve as predetermined breaking points that, when exposed, help to target misfolded or surplus proteins for degradation, thereby maintaining a healthy membrane proteome.

A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines.

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and ß subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate ß subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone-client complexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:ß assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones.

Intramembrane client recognition potentiates the chaperone functions of calnexin.

One-third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar-binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin-based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.

Biogenesis and engineering of interleukin 12 family cytokines.

Interleukin 12 (IL-12) family cytokines are secreted proteins that regulate immune responses. Each family member is a heterodimer and nature uses shared building blocks to assemble the functionally distinct IL-12 cytokines. In recent years we have gained insights into the molecular principles and cellular regulation of IL-12 family biogenesis. For each of the family members, generally one subunit depends on its partner to acquire its native structure and be secreted from immune cells. If unpaired, molecular chaperones retain these subunits in cells. This allows cells to regulate and control secretion of the highly potent IL-12 family cytokines. Molecular insights gained into IL-12 family biogenesis, structure, and function now allow us to engineer IL-12 family cytokines to develop novel immunotherapeutic approaches.

IL-30† (IL-27A): a familiar stranger in immunity, inflammation, and cancer.

Over the years, interleukin (IL)-27 has received much attention because of its highly divergent, sometimes even opposing, functions in immunity. IL-30, the p28 subunit that forms IL-27 together with Ebi3 and is also known as IL-27p28 or IL-27A, has been considered a surrogate to represent IL-27. However, it was later discovered that IL-30 can form complexes with other protein subunits, potentially leading to overlapping or discrete functions. Furthermore, there is emerging evidence that IL-30 itself may perform immunomodulatory functions independent of Ebi3 or other binding partners and that IL-30 production is strongly associated with certain cancers in humans. In this review, we will discuss the biology of IL-30 and other IL-30-associated cytokines and their functions in inflammation and cancer.

Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

Quality control of mislocalized and orphan proteins.

A healthy and functional proteome is essential to cell physiology. However, this is constantly being challenged as most steps of protein metabolism are error-prone and changes in the physico-chemical environment can affect protein structure and function, thereby disrupting proteome homeostasis. Among a variety of potential mistakes, proteins can be targeted to incorrect compartments or subunits of protein complexes may fail to assemble properly with their partners, resulting in the formation of mislocalized and orphan proteins, respectively. Quality control systems are in place to handle these aberrant proteins, and to minimize their detrimental impact on cellular functions. Here, we discuss recent findings on quality control mechanisms handling mislocalized and orphan proteins. We highlight common principles involved in their recognition and summarize how accumulation of these aberrant molecules is associated with aging and disease.

Fluorescent palladium(II) and platinum(II) NHC/1,2,3-triazole complexes: antiproliferative activity and selectivity against cancer cells.

Fluorescent Pd(ii) and Pt(ii) complexes bearing 4-methylene-7-methoxycoumarin (MMC) and 2,6-diispropylphenyl (Dipp) substituted NHC/1,2,3-triazole hybrid ligands are described. Depending on the reaction conditions two different ligand coordination modes are observed, i.e., bidentate solely coordinating via NHCs or tetradentate coordinating via NHCs and 1,2,3-triazoles. All Dipp substituted complexes show antiproliferative activity against cervix (HeLa) and breast (MCF-7) human carcinoma cells. The activity significantly depends on the coordination mode, with the tetradentate motif being notably more effective (HeLa: IC50 = 3.9 µM to 4.7 µM; MCF-7: IC50 = 2.07 µM to 2.35 µM). Amongst the MMC series, only the Pd(ii) complex featuring the bidentate coordination mode is active against HeLa (IC50 = 6.1 µM). In contrast to its structurally related Dipp derivative (SI = 0.6), it shows a high selectivity for HeLa (SI > 16) compared to healthy skin cells (HaCaT). According to fluorescence microscopy, this compound is presumably located in late endosomes or lysosomes.

Influence of glycosylation on IL-12 family cytokine biogenesis and function.

The interleukin 12 (IL-12) family of cytokines regulates T cell functions and is key for the orchestration of immune responses. Each heterodimeric IL-12 family member is a glycoprotein. However, the impact of glycosylation on biogenesis and function of the different family members has remained incompletely defined. Here, we identify glycosylation sites within human IL-12 family subunits that become modified upon secretion. Building on these insights, we show that glycosylation is dispensable for secretion of human IL-12 family cytokines except for IL-35. Furthermore, our data show that glycosylation differentially influences IL-12 family cytokine functionality, with IL-27 being most strongly affected. Taken together, our study provides a comprehensive analysis of how glycosylation affects biogenesis and function of a key human cytokine family and provides the basis for selectively modulating their secretion via targeting glycosylation.

Glycosaminoglycans bind human IL-27 and regulate its activity.

IL-27 is a cytokine of the IL-12 family, composed of EBI3 and IL-27p28. IL-27 regulates immune responses and also other physiological processes including hematopoiesis, angiogenesis, and bone formation. Its receptor, composed of IL-27Rα and gp130, activates the STAT pathway. Here, we show that different glycosaminoglycans (GAGs) modulate human IL-27 activity in vitro. We find that soluble heparin and heparan sulfate efficiently inhibit human IL-27 activity as shown by decreased STAT signaling and downstream biological effects. In contrast, membrane-bound heparan sulfate seems to positively regulate IL-27 activity. Our biochemical studies demonstrate that soluble GAGs directly bind to human IL-27, consistent with in silico analyses, and prevent its binding to IL-27Rα. Although murine IL-27 also bound to GAGs in vitro, its activity was less efficiently inhibited by soluble GAGs. Lastly, we show that two heparin-derivatives, low molecular weight heparin and fondaparinux, that like unfractionated heparin are used in clinics, had weaker or no effect on human IL-27 activity. Together, our data identify GAGs as new players in the regulation of human IL-27 activity that might act under physiological conditions and may also have a clinical impact in heparin-treated patients.

An anti-inflammatory eicosanoid switch mediates the suppression of type-2 inflammation by helminth larval products.

Eicosanoids are key mediators of type-2 inflammation, e.g., in allergy and asthma. Helminth products have been suggested as remedies against inflammatory diseases, but their effects on eicosanoids are unknown. Here, we show that larval products of the helminth Heligmosomoides polygyrus bakeri (HpbE), known to modulate type-2 responses, trigger a broad anti-inflammatory eicosanoid shift by suppressing the 5-lipoxygenase pathway, but inducing the cyclooxygenase (COX) pathway. In human macrophages and granulocytes, the HpbE-driven induction of the COX pathway resulted in the production of anti-inflammatory mediators [e.g., prostaglandin E2 (PGE2) and IL-10] and suppressed chemotaxis. HpbE also abrogated the chemotaxis of granulocytes from patients suffering from aspirin-exacerbated respiratory disease (AERD), a severe type-2 inflammatory condition. Intranasal treatment with HpbE extract attenuated allergic airway inflammation in mice, and intranasal transfer of HpbE-conditioned macrophages led to reduced airway eosinophilia in a COX/PGE2-dependent fashion. The induction of regulatory mediators in macrophages depended on p38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor-1α (HIF-1α), and Hpb glutamate dehydrogenase (GDH), which we identify as a major immunoregulatory protein in HpbE Hpb GDH activity was required for anti-inflammatory effects of HpbE in macrophages, and local administration of recombinant Hpb GDH to the airways abrogated allergic airway inflammation in mice. Thus, a metabolic enzyme present in helminth larvae can suppress type-2 inflammation by inducing an anti-inflammatory eicosanoid switch, which has important implications for the therapy of allergy and asthma.

In case of stress, hold tight: phosphorylation switches PDI from an oxidoreductase to a holdase, tuning ER proteostasis.

Eukaryotic cells have evolved multiple responses that allow endoplasmic reticulum (ER) homeostasis to be maintained even in the face of acute or chronic stresses. In this issue, Yu et al (2020) describe how site-specific phosphorylation switches protein disulfide isomerase (PDI) from a folding enzyme to a holdase chaperone which regulates ER stress responses, thus highlighting PDI as a key player in ER homeostasis.

Site-Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover.

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio-orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence-based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse-chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.

Structural Insight into IAPP-Derived Amyloid Inhibitors and Their Mechanism of Action.

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aß (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aß amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a ß-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid-liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aß40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.