TNF-α influences the lateral dynamics of TNF receptor I in living cells

In mammalian cells, inflammation is mainly mediated by the binding of tumor necrosis factor alpha to tumor necrosis factor receptor 1. In this study, we investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy. Our single-molecule data indicates the presence of tumor necrosis factor receptor 1 with different mobilities in the plasma membrane, suggesting different molecular organizations. Cholesterol depletion led to a decrease of slow receptor species and a strong increase in the average diffusion coefficient. Moreover, as a consequence of tumor necrosis factor-alpha treatment, the mean diffusion coefficient moderately increased while its distribution narrowed. Based on our observation, we propose a refined mechanism on the structural arrangement and activation of tumor necrosis factor receptor 1 in the plasma membrane.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

Werner syndrome protein works as a dimer for unwinding and replication fork regression

The determination of the oligomeric state of functional enzymes is essential for the mechanistic understanding of their catalytic activities. RecQ helicases have diverse biochemical activities, but it is still unclear how their activities are related to their oligomeric states. We use single-molecule multi-color fluorescence imaging to determine the oligomeric states of Werner syndrome protein (WRN) during its unwinding and replication fork regression activities. We reveal that WRN binds to a forked DNA as a dimer, and unwinds it without any change of its oligomeric state. In contrast, WRN binds to a replication fork as a tetramer, and is dimerized during activation of replication fork regression. By selectively inhibiting the helicase activity of WRN on specific strands, we reveal how the active dimers of WRN distinctly use the energy of ATP hydrolysis for repetitive unwinding and replication fork regression.     

Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.     

Macromolecular mass spectrometry and electron microscopy as complementary tools for investigation of the heterogeneity of bacteriophage portal assemblies

The success of electron-cryo microscopy (cryo-EM) and image reconstruction of cyclic oligomers, such as the viral and bacteriophage portals, depends on the accurate knowledge of their order of symmetry. A number of statistical methods of image analysis address this problem, but often do not provide unambiguous results. Direct measurement of the oligomeric state of multisubunit protein assemblies is difficult when the number of subunits is large and one subunit renders only a small increment to the full size of the oligomer. Moreover, when mixtures of different stochiometries are present techniques such as analytical centrifugation or size-exclusion chromatography are also less helpful. Here, we use electrospray ionization mass spectrometry to directly determine the oligomeric states of the in vitro assembled portal oligomers of the phages P22, Phi-29 and SPP1, which range in mass from 430 kDa to about 1 million Da. Our data unambiguously reveal that the oligomeric states of Phi-29 and SPP1 portals were 12 and 13, respectively, in good agreement with crystallographic and electron microscopy data. However, in vitro assembled P22 portals were a mixture of 11- and 12-mer species in an approximate ratio of 2:1, respectively. A subsequent reference-free alignment of electron microscopy images of the P22 portal confirmed this mixture of oligomeric states. We conclude that macromolecular mass spectrometry is a valuable tool in structural biology that can aide in the determination of oligomeric states and symmetry of assemblies, providing a good starting point for improved image analysis of cryo-EM data.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

Cell Fate Decisions Regulated by K63 Ubiquitination of Tumor Necrosis Factor Receptor 1

Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival. Downmodulation of RNF8 prevented the ubiquitination of TNF-R1, blocked the internalization of the receptor, prevented the recruitment of the death-inducing signaling complex and the activation of caspase-8 and caspase-3/7, and reduced apoptotic cell death. Conversely, recruitment of the adaptor proteins TRADD, TRAF2, and RIP1 to TNF-R1, as well as activation of NF-κB, was unimpeded and cell growth and proliferation were significantly enhanced in RNF8-deficient cells. Thus, K63 ubiquitination of TNF-R1 can be sensed as a new level of regulation of TNF-R1 signaling at the earliest stage after ligand binding.     

Involvement of an Asn/Val cleavage site in the production of a soluble form of a human tumor necrosis factor (TNF) receptor. Site-directed mutagenesis of a putative cleavage site in the p55 TNF receptor chain.

Soluble forms of receptors for tumor necrosis factor (TNF) might be important for regulating the actions of TNF. Site-directed in vitro mutagenesis was employed to examine the processing of the p55 tumor necrosis factor receptor chain (TNF-R55) into soluble TNF-binding protein (TNF-R55-BP). An Asn/Val sequence close to the transmembrane region in TNF-R55 was indicated as a putative cleavage site for proteolytic processing. By mutagenesis, Asn and Val were replaced with Gly and Ala, respectively. Expression in Chinese hamster ovary (CHO) cells resulted in identical binding of ligand to mutated receptors as compared to receptors not subjected to mutagenesis. Turnover rates of receptors as judged by disappearance of TNF binding capacity after inhibition of de novo protein synthesis and downregulation in response to incubation with phorbol esters were also identical between wild-type and mutated receptors. However, mutations of the cleavage site resulted in a decreased spontaneous and phorbol ester-induced release of soluble receptor (TNF-R55-BP) which was almost abolished when both Asn and Val were mutated. Our results clearly demonstrate the importance of an Asn/Val sequence for proteolytic processing of the TNF-R55 into soluble TNF-R55-BP and indicate that phorbol ester-induced downregulation of the TNF-R55 may be dissociated from proteolytic cleavage of the receptor.     

The role of tyrosine sulfation in the dimerization of the CXCR4:SDF‐1 complex

Oligomerization of G protein‐coupled receptors is a recognized mode of regulation of receptor activities, with alternate oligomeric states resulting in different signaling functions. The CXCR4 chemokine receptor is a G protein‐coupled receptor that is post‐translationally modified by tyrosine sulfation at three sites on its N‐terminus (Y7, Y12, Y21), leading to enhanced affinity for its ligand, stromal cell derived factor (SDF‐1, also called CXCL12). The complex has been implicated in cancer metastasis and is a therapeutic target in cancer treatment. Using molecular dynamics simulation of NMR‐derived structures of the CXCR4 N‐terminus in complex with SDF‐1, and calculations of electrostatic binding energies for these complexes, we address the role of tyrosine sulfation in this complex. Our results show that sulfation stabilizes the dimeric state of the CXCR4:SDF‐1 complex through hydrogen bonding across the dimer interface, conformational changes in residues at the dimer interface, and an enhancement in electrostatic binding energies associated with dimerization. These findings suggest a mechanism through which post‐translational modifications such as tyrosine sulfation might regulate downstream function through modulation of the oligomeric state of the modified system.     

Differential TNF-signaling in chronic inflammatory disorders

TNF-alpha is a pleiotropic cytokine with strong proinflammatory and immunomodulatory properties. TNF-alpha plays a critical role in many acute or chronic inflammatory diseases and anti-TNF-strategies have proven to be clinically effective. Two TNF-specific cell surface receptors TNF-R1 and TNF-R2 have been identified and the function of these receptors and the downstream intracellular signal transduction pathways have been extensively studied in vitro. For a long time TNF-R1 was considered to be the predominant mediator of TNF-signaling, whereas TNF-R2 was ascribed only auxilliary function. However, there is increasing clinical and experimental evidence for an important independent role of p80 signaling in chronic inflammatory conditions. It is conceivable that the multiple TNF-mediated chronic inflammatory disorders differ in terms of the ligand form (soluble TNF-alpha versus membrane bound TNF-alpha), the receptor (TNF-R1 versus TNF-R2) and the downstream signaling cascades utilized. The elucidation of the specific characteristics of TNF-signaling in distinct inflammatory disorders will lead to a better understanding ot the pathogenesis of these diseases and will be the basis for the development of more specific and more efficient therapeutic approaches.     

Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization

Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.     

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in two-dimensional or three-dimensional single-molecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions.     

Discoidin domain receptors: Micro insights into macro assemblies

Assembly of cell-surface receptors into specific oligomeric states and/or clusters before and after ligand binding is an important feature governing their biological function. Receptor oligomerization can be mediated by specific domains of the receptor, ligand binding, configurational changes or other interacting molecules. In this review we summarize our understanding of the oligomeric state of discoidin domain receptors (DDR1 and DDR2), which belong to the receptor tyrosine kinase family (RTK). DDRs form an interesting system from an oligomerization perspective as their ligand collagen(s) can also undergo supramolecular assembly to form fibrils. Even though DDR1 and DDR2 differ in the domains responsible to form ligand-free dimers they share similarities in binding to soluble, monomeric collagen. However, only DDR1b forms globular clusters in response to monomeric collagen and not DDR2. Interestingly, both DDR1 and DDR2 are assembled into linear clusters by the collagen fibril. Formation of these clusters is important for receptor phosphorylation and is mediated in part by other membrane components. We summarize how the oligomeric status of DDRs shares similarities with other members of the RTK family and with collagen receptors. Unraveling the multiple macro-molecular configurations adopted by this receptor-ligand pair can provide novel insights into the intricacies of cell-matrix interactions.     

Site-directed cross-linking. Establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis

Cysteine residues introduced at specific locations in the aspartate receptor of Salmonella typhimurium provide anchor points for cross-linking and serve as chemical markers for structural studies of this oligomeric receptor. These markers have been used to measure the rate of subunit exchange between oligomeric receptors and to show that ligand binding inhibits this exchange. The cysteine-containing receptors can be oxidatively cross-linked to completion within the oligomeric receptor, indicating that the receptor has an even number of subunits. Based on this observation, a technique has been developed that can be used to determine the oligomeric structure of proteins under a variety of experimental conditions. The technique involves the measurement of the effect of dilution by "cysteineless" receptor subunits on cross-linking and reveals that the aspartate receptor is dimeric in detergent solution, in a mixed-micelle system, and in reconstituted membrane vesicles. Binding of aspartate does not change the oligomeric structure of the receptor, indicating that transmembrane signaling occurs within an oligomeric receptor of constant size.     

Oligomerization Interface of RAGE Receptor Revealed by MS-Monitored Hydrogen Deuterium Exchange

Activation of the receptor for advanced glycation end products (RAGE) leads to a chronic proinflammatory signal, affecting patients with a variety of diseases. Potentially beneficial modification of RAGE activity requires understanding the signal transduction mechanism at the molecular level. The ligand binding domain is structurally uncoupled from the cytoplasmic domain, suggesting receptor oligomerization is a requirement for receptor activation. In this study, we used hydrogen-deuterium exchange and mass spectrometry to map structural differences between the monomeric and oligomeric forms of RAGE. Our results indicated the presence of a region shielded from exchange in the oligomeric form of RAGE and led to the identification of a new oligomerization interface localized at the linker region between domains C1 and C2. Based on this finding, a model of a RAGE dimer and higher oligomeric state was constructed.     

Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells

Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique—spatial intensity distribution analysis (SpIDA)—that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.     

Prolonged allograft survival in TNF receptor 1-deficient recipients is due to immunoregulatory effects, not to inhibition of direct antigraft cytotoxicity.

TNF-alpha and lymphotoxin (LT)alpha have been shown to be important mediators of allograft rejection. TNF-R1 is the principal receptor for both molecules. Mice with targeted genetic deletions of TNF-R1 demonstrate normal development of T and B lymphocytes but exhibit functional defects in immune responses. However, the role of TNF-R1-mediated signaling in solid organ transplant rejection has not been defined. To investigate this question, we performed vascularized heterotopic allogeneic cardiac transplants in TNF-R1-deficient (TNF-R1(-/-)) and wild-type mice. Because all allografts in our protocol expressed TNF-R1, direct antigraft effects of TNF-alpha and LTalpha were not prevented. However, immunoregulatory effects on recipient inflammatory cells by TNF-R1 engagement was eliminated in TNF-R1(-/-) recipients. In our study, cardiac allograft survival was significantly prolonged in TNF-R1(-/-) recipients. Despite this prolonged allograft survival, we detected increased levels of CD8 T cell markers in allografts from TNF-R1(-/-) recipients, suggesting that effector functions, but not T cell recruitment, were blocked. We also demonstrated the inhibition of multiple chemokines and cytokines in allografts from TNF-R1(-/-) recipients including RANTES, IFN-inducible protein-10, lymphotactin, and IL-1R antagonist, as well as altered levels of chemokine receptors. We correlated gene expression with the physiologic process of allograft rejection using self-organizing maps and identified distinct patterns of gene expression in allografts from TNF-R1(-/-) recipients. These findings indicate that in our experimental system TNF-alpha and LTalpha exert profound immunoregulatory effects through TNF-R1.     

Morphology of epidermoid carcinoma A431 cells spread on immobilized ligands

Cell interaction with extracellular matrix is a multi-step process characterized by cell attachment to substrata with subsequent cell spreading accompanied by actin cytoskeleton and cellular membrane receptor reorganization. It has been shown elsewhere that epidermoid carcinoma A431 cells, spread on solid substrata coated with fibronectin, laminin-2/4 or antibodies to EGF receptor, form specific actin filament structures typical for each particular ligand. Here quantitative analysis of heterogeneous A431 cell population spread on the above ligands has been reported. Cells were subdivided into morphological classes, according to their shape and actin filament structure, and the relationship among classes under various experimental conditions were quantitatively estimated for every ligand. We studied the influence of cell detachment pattern, short-term and long-term starvation, and cell incubation in suspended state in the medium before plating on the cell population composition. It was possible to recognize the modal morphological class of cells with typical actin cytoskeleton structure dominating for the ligand in the population. Long-term starvation and incubation in suspension before cell spreading are considered as the crucial experimental parameters leading to dramatic changes in cell population.     

LMP1, a viral relative of the TNF receptor family,signals principally from intracellular compartments

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has significantly reduced localization at the plasma membrane yet signals as efficiently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.