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The protein periostin is a matricellular protein that is expressed in connective tissue. It is composed of five globular domains arranged in an elongated structure with an extensive disordered C-terminal tail. Periostin contains 11 cysteine residues, of which one is unpaired and the rest form five intramolecular disulfide bonds. Periostin plays an important role during wound healing and is also involved in driving the inflammatory state in atopic diseases. This study provides a comprehensive biochemical characterization of periostin in human skin and in dermal and pulmonary fibroblasts in vitro. Through the application of Western blotting, co-immunoprecipitation, and LC-MS/MS, we show for the first time that periostin is a disulfide-bonded homodimer and engages in a novel disulfide-bonded complex with fibronectin both in vivoand in vitro. This inherent characteristic of periostin holds the potential to redefine our approach to exploring and understanding its functional role in future research endeavors.

Nanoparticles (NPs) can modulate protein aggregation and fibril formation in the context of amyloid diseases. Understanding the mechanism of this action remains a critical next step in developing nanomedicines for the treatment or prevention of Parkinson’s disease. α-Synuclein (α-Syn) can undergo interactions of different strength with nanoparticles, and these interactions can be prevented by the presence of a protein corona (PC) acquired during the exposure of NPs to serum proteins. Here, we develop a method to attach the PC irreversibly to the NPs, which enables us to study in detail the interaction of α-Syn and polyethylenimine-coated carboxyl-modified polystyrene NPs (PsNPs-PEI) and the role of the dynamics of the interactions. Analysis of the kinetics of fibril formation reveals that the NPs surface promotes the primary nucleation step of amyloid fibril formation without significantly affecting the elongation and fragmentation steps or the final equilibrium. Furthermore, the results show that even though α-Syn can access the surface of NPs that are precoated with a PC, due to the dynamic nature of the PC proteins, the PC nevertheless reduces the acceleratoring effect of the NPs. This effect is likely to be caused by reducing the overall amount of weakly interacting α-Syn molecules on the NP surface and the access of further α-Syn required for fibril elongation. Our experimental approach provides microscopic insight into how serum proteins can modulate the complex interplay between NPs and amyloid proteins.

Most proteins in the α-macroglobulin (αM) superfamily contain reactive thiol esters that are required for their biological function. Here, we have characterized the human α2-macroglobulin (A2M) and complement component C3 mutants A2M Q975C and C3 Q1013C, which replace the CGEQ thiol ester motifs of the original proteins with the disulfide-forming sequence CGEC. Mass spectrometry showed that the intended disulfide was formed in both proteins. The correct folding and native conformation of A2M Q975C were shown by its assembly to a tetramer, an initially slow electrophoretic mobility with a demonstrable conformational collapse induced by proteolysis, functional protease trapping, and conformation-dependent interactions with low-density lipoprotein receptor-related protein 1. However, A2M Q975C had a decreased capacity to inhibit trypsin and was more susceptible to cleavage by trypsin or thermolysin when compared to wild-type A2M. C3 Q1013C also folded correctly and was initially in a native conformation, as demonstrated by its cation exchange elution profile, electrophoretic mobility, and interaction with complement factor B, although it assumed a conformation that was distinct from native C3, C3b, or C3(H2O) when cleaved by trypsin. These results demonstrate that disulfides can substitute thiol esters and maintain the native conformations of A2M and C3. Additionally, they indicate that proteolysis is not the sole factor in the conformational changes of A2M and C3 and that thiol ester lysis also plays a role.

Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, “non-self” incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivofate of intravenously injected 70 nm SiO2nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Aggregation of the Amyloid β peptide into amyloid fibrils is closely related to development of Alzheimer’s disease. Many small aromatic compounds have been found to act as inhibitors of fibril formation, and have inspired the search for new drug candidates. However, the detailed mechanisms of inhibition are largely unknown. In this study, we have examined in detail the binding of the fibril-formation inhibitor Congo Red (CR) to monomeric Aβ1−40using a combination of 1D, 2D, saturation transfer difference, and diffusion NMR, as well as dynamic light scattering experiments. Our results show that CR binds to the fibril forming stretches of Aβ1−40monomers, and that complex formation occurs in two steps: An initial 1:1 CR:Aβ1−40complex is formed by a relatively strong interaction (Kd≈ 5 μM), and a 2:1 complex is formed by binding another CR molecule in a subsequent weaker binding step (Kd≈ 300 μM). The size of these complexes is comparable to that of Aβ1−40alone. The existence of two different complexes might explain the contradictory reports regarding the inhibitory effects of CR on the fibril-formation process.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

The intrinsically disordered protein α-synuclein (aSN) forms insoluble aggregates in the brains of Parkinson’s disease (PD) patients. Cytotoxicity is attributed to a soluble aSN oligomeric species that permeabilizes membranes significantly more than monomers and fibrils. In humans, the A53T mutation induces early onset PD and increases the level of aSN oligomerization and fibrillation propensity, but Thr53 occurs naturally in aSNs of most animals. We compared aSNs from elephant, bowhead whale, and pig with human aSN. While all three animal aSNs showed significantly weakened fibrillation, elephant aSN formed much more oligomer, and pig aSN much less, than human aSN did. However, all animal aSN oligomers showed weakened permeabilization toward anionic lipid vesicles, indicative of decreased cytotoxicity. These animal aSNs share three substitutions compared to human aSN: A53T, G68E, and V95G. We analyzed aggregation and membrane binding of all eight mutants combining these three mutations. While the G68E mutation is particularly important in weakening fibrillation and possible toxicity, the strongest effect is seen when all three mutations are present. Thus, a small number of mutations can significantly decrease aSN toxicity.

Protein conjugates of high heterogeneity may contain species with significantly different biological properties, and as a consequence, the focus on methods for production of conjugates of higher quality has increased. Here, we demonstrate an efficient and generic approach for the modification of metal-binding proteins with biocompatible chemical handles without the need for genetic modifications. Affinity-guided small-molecule probes are developed for direct conjugation to off-the-shelf proteins and for installing different chemical handles on the protein surface. While purification of protein conjugates obtained by small molecule conjugation is troublesome, the affinity motifs of the probes presented here allow for purification of the conjugates. The versatility of the probes is demonstrated by conjugation to several His-tagged and natural metal-binding proteins, including the efficient and area-selective conjugation to three therapeutically relevant antibodies.

Mutations in the transforming growth factor β-induced protein (TGFBIp) cause phenotypically diverse corneal dystrophies, where protein aggregation in the cornea leads to severe visual impairment. Previous studies have shown a relationship between mutant-specific corneal dystrophy phenotypes and the thermodynamic stability of TGFBIp. Using liquid chromatography–tandem mass spectrometry and nuclear magnetic resonance (NMR), we investigated correlations between the structural integrity of disease-related mutants of the fourth FAS1 domain (FAS1-4) and deamidation of TGFBIp residue Asn622. We observed a high rate of Asn622 deamidation in the A546D and A546D/P551Q FAS1-4 mutants that were both largely unstructured as determined by NMR. Conversely, the more structurally organized A546T and V624M FAS1-4 mutants had reduced deamidation rates, suggesting that a folded and stable FAS1-4 domain precludes Asn622 deamidation. Wild-type, R555Q, and R555W FAS1-4 mutants displayed very slow deamidation, which agrees with their similar and ordered NMR structures, where Asn622 is in a locked conformation. We confirmed the FAS1-4 mutational effect on deamidation rates in full-length TGFBIp mutants and found a similar ranking compared to that of the FAS1-4 domain alone. Consequently, the deamidation rate of Asn622 can be used to predict the structural effect of the many destabilizing and/or stabilizing mutations reported for TGFBIp. In addition, the deamidation of Asn622 may influence the pathophysiology of TGFBIp-induced corneal dystrophies.

Many proteins and peptides self-associate into highly ordered and structurally similar amyloid cross-βaggregates. This fibrillation is critically dependent on properties of the protein and the surrounding environment that alter kinetic and thermodynamic equilibria. Here, we report on dominating surface and solution effects on the fibrillogenic behavior and amyloid assembly of the C-36 peptide, a circulating bioactive peptide from the α1-antitrypsin serine protease inhibitor. C-36 converts from an unstructured peptide to mature amyloid twisted-ribbon fibrils over a few hours when incubated on polystyrene plates under physiological conditions through a pathway dominated by surface-enhanced nucleation. In contrast, in plates with nonbinding surfaces, slow bulk nucleation takes precedence over surface catalysis and leads to fibrillar polymorphism. Fibrillation is strongly ion-sensitive, underlining the interplay between hydrophilic and hydrophobic forces in molecular self-assembly. The addition of exogenous surfaces in the form of silica glass beads and polyanionic heparin molecules potently seeds the amyloid conversion process. In particular, heparin acts as an interacting template that rapidly forces β-sheet aggregation of C-36 to distinct amyloid species within minutes and leads to a more homogeneous fibril population according to solid-state NMR analysis. Heparin’s template effect highlights its role in amyloid seeding and homogeneous self-assembly, which applies both in vitro and in vivo, where glycosaminoglycans are strongly associated with amyloid deposits. Our study illustrates the versatile thermodynamic landscape of amyloid formation and highlights how different experimental conditions direct C-36 into distinct macromolecular structures.

The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca2+concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

Protease inhibition by metastable serine protease inhibitors (serpins) is mediated by one of the largest functional intradomain conformational changes known in biology. In this extensive structural rearrangement, protease–serpin complex formation triggers cleavage of the serpin reactive center loop (RCL), its subsequent insertion into central β-sheet A, and covalent trapping of the target protease. In this study, we present the first detailed accelerated molecular dynamics simulation of the insertion of the fully cleaved RCL in α-1-antitrypsin (α1AT), the archetypal member of the family of human serpins. Our results reveal internal water pathways that allow the initial incorporation of side chains of RCL residues into the protein interior. We observed structural plasticity of the helix F (hF) element that blocks the RCL path in the native state, which is in excellent agreement with previous experimental reports. Furthermore, the simulation suggested a novel role of hF and the connected turn (thFs3A) as chaperones that support the insertion process by reducing the conformational space available to the RCL. Transient electrostatic interactions of RCL residues potentially fine-tune the serpin inhibitory activity. On the basis of our simulation, we generated the α1AT mutants K168E, E346K, and K168E/E346K and analyzed their inhibitory activity along with their intrinsic stability and heat-induced polymerization. Remarkably, the E346K mutation exhibited enhanced inhibitory activity along with an increased rate of premature structural collapse (polymerization), suggesting a significant role of E346 in the gatekeeping of the strain in the metastable native state.

Infection by the opportunistic pathogen Pseudomonas aeruginosa(PA) is accompanied by the secretion of virulence factors such as the secondary metabolite rhamnolipid (RL) as well as an array of bacterial enzymes, including the peptidase elastase. The human immune system tries to counter this via defensive proteins such as lysozyme (HLZ). HLZ targets the bacterial cell wall but may also have other antimicrobial activities. The enzyme contains four disulfide bonds and shows high thermodynamic stability and resistance to proteolytic attack. Here we show that RL promotes HLZ degradation by several unrelated peptidases, including the PA elastase and human peptidases. This occurs although RL does not by itself denature HLZ. Nevertheless, RL binds in a sufficiently high stoichiometry (8:1 RL:HLZ) to neutralize the highly cationic surface of HLZ. The initial cleavage sites agree well with the domain boundaries of HLZ. Thus, binding of RL to native HLZ may be sufficient to allow proteolytic attack at slightly exposed sites on the protein, leading to subsequent degradation. Furthermore, biofilms of RL-producing strains of PA are protected better against high concentrations of HLZ than RL-free PA strains are. We conclude that pathogen-produced RL may weaken host defenses by facilitating degradation of key host proteins.

Biomolecule decoration of nanoparticles provides a corona that modulates how the nanoparticles interact with biological milieus. The corona composition has proved to reflect the differences in the repertoire of proteins to which the nanoparticles are exposed, and as a result the same nanoparticles can acquire a differential biological identity. Here we examined whether a unique biological identity acquired from sex-specific protein repertoires could alter the degree of nanoparticle uptake by cognate immune cells. We chose zebrafish as a model species of which blood plasma is sexually contrasted by the unique presence/absence of the egg yolk precursor protein vitellogenin. Sex-specific protein coronas were thus formed around 70 nm SiO2nanoparticles using female/male blood plasma from zebrafish or fetal bovine serum as a non-native reference. In contrast to protein coronas formed of male blood plasma, a “female” biological identity of the nanoparticles was represented by prevailing contribution of vitellogenins to the corona proteome. We then exposed zebrafish blood cells to the three types of pre-formed nanoparticle–protein complexes and compared nanoparticle uptake using flow cytometry. Lymphoid and myeloid populations of the blood cells preferentially accumulated the nanoparticles with a female biological identity, irrespective of the sex of the fish from which the cells were obtained. The concept of repertoire differences in the corona proteome therefore deserves further attention, as various factors such as sex-specific biological conditions exemplified in this study could alter the nanoparticle–cell interactions.

Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH2-terminal cysteine-rich domain (CRD) annotated as an emilin (EMI) domain and four fasciclin-1 (FAS1-1–FAS1-4) domains. Mutations in the gene cause corneal dystrophies, a group of debilitating protein misfolding diseases that lead to severe visual impairment. Previous studies have shown that TGFBIp in the cornea is cross-linked to type XII collagen through a reducible bond. TGFBIp contains 11 cysteine residues and is thus able to form five intramolecule disulfide bonds, leaving a single cysteine residue available for the collagen cross-link. The structures of TGFBIp and its homologues are unknown. We here present the disulfide bridge pattern of TGFBIp, which was determined by generating specific peptides. These were separated by ion exchange followed by reversed-phase high-performance liquid chromatography and analyzed by mass spectrometry and Edman degradation. The NH2-terminal CRD contains six cysteine residues, and one of these (Cys65) was identified as the candidate for the reducible cross-link between TGFBIp and type XII collagen. In addition, the CRD contained two intradomain disulfide bridges (Cys49–Cys85 and Cys84–Cys97) and one interdomain disulfide bridge to FAS1-2 (Cys74–Cys339). Significantly, this arrangement violates the predicted disulfide bridge pattern of an EMI domain. The cysteine residues in FAS1-3 (Cys473 and Cys478) were shown to form an intradomain disulfide bridge. Finally, an interdomain disulfide bridge between FAS1-1 and FAS1-2 (Cys214–Cys317) was identified. The interdomain disulfide bonds indicate that the NH2terminus of TGFBIp (CRD, FAS1-1, and FAS1-2) adopts a compact globular fold, leaving FAS1-3 and FAS1-4 exposed.

Carbamylation of lysine residues and protein N-termini is an ubiquitous, non-enzymatic post-translational modification. Carbamylation at sites of inflammation is due to cyanate formation during the neutrophil oxidative burst and may target lysine residues within the antimicrobial peptide LL-37. The bactericidal and immunomodulatory properties of LL-37 depend on its secondary structure and cationic nature, which are conferred by arginine and lysine residues. Therefore, carbamylation may affect the biological functions of LL-37. The present study examined the kinetics and pattern of LL-37 carbamylation to investigate how this modification affects the bactericidal, cytotoxic and immunomodulatory function of the peptide. The results indicated that LL-37 undergoes rapid modification in the presence of physiological concentrations of cyanate, yielding a spectrum of diverse carbamylated peptides. Mass spectrometry analyses revealed that the N-terminal amino group of Leu-1 was highly reactive and was modified almost instantly by cyanate to generate the predominant form of the modified peptide, named LL-37C1. This was followed by the sequential carbamylation of Lys-8, Lys-12, and Lys-15 to yield LL-37C8, and Lys-15 to yield LL-37C12,15. Carbamylation had profound and diverse effects on the structure and biological properties of LL-37. In some cases, anti-inflammatory LL-37 was rapidly converted to pro-inflammatory LL-37.

Human periostin is a 78–91 kDa matricellular protein implicated in extracellular matrix remodeling, tumor development, metastasis, and inflammatory diseases like atopic dermatitis, psoriasis, and asthma. The protein consists of six domains, including an N-terminal Cys-rich CROPT domain, four fasciclin-1 domains, and a C-terminal domain. The exons encoding the C-terminal domain may be alternatively spliced by shuffling four exons, generating ten variants of unknown function. Here, we investigate the structure and interactome of the full-length variant of the C-terminal domain with no exons spliced out. The structural analysis showed that the C-terminal domain lacked a tertiary structure and was intrinsically disordered. In addition, we show that the motif responsible for heparin-binding is in the conserved very C-terminal part of periostin. Pull-down confirmed three known interaction partners and identified an additional 140 proteins, among which nine previously have been implicated in atopic dermatitis. Based on our findings, we suggest that the C-terminal domain of periostin facilitates interactions between connective tissue components in concert with the four fasciclin domains.