Hydrophobic CDR3 residues promote the development of self-reactive T cells.

Studies of individual T cell antigen receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, the general rules that can be used to predict whether TCRs are self-reactive have not been fully elucidated. Here we found that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the complementarity-determining region CDR3ß robustly promoted the development of self-reactive TCRs. This property was found irrespective of the member of the ß-chain variable region (Vß) family present in the TCR or the length of the CDR3ß. An index based on these findings distinguished Vß2(+), Vß6(+) and Vß8.2(+) regulatory T cells from conventional T cells and also distinguished CD4(+) T cells selected by the major histocompatibility complex (MHC) class II molecule I-A(g7) (associated with the development of type 1 diabetes in NOD mice) from those selected by a non-autoimmunity-promoting MHC class II molecule I-A(b). Our results provide a means for distinguishing normal T cell repertoires versus autoimmunity-prone T cell repertoires.

Role of linear ubiquitination in inflammatory responses and tissue homeostasis.

Polyubiquitination is a post-translational modification involved in a wide range of immunological events, including inflammatory responses, immune cell differentiation, and development of inflammatory diseases. The versatile functions of polyubiquitination are based on different types of ubiquitin linkage, which enable various UBD (ubiquitin binding domain)-containing adaptor proteins to associate and induce distinct biological outputs. A unique and atypical type of polyubiquitin chain comprising a conjugation between the N-terminal methionine of the proximal ubiquitin moiety and the C-terminal glycine of the distal ubiquitin moiety, referred to as a linear or M1-linked ubiquitin chain, has been studied exclusively within the field of immunology because it is distinct from other polyubiquitin forms: linear ubiquitin chains are generated predominantly by various inflammatory stimulants, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and act as a critical modulator of transient and optimal signal transduction. Moreover, accumulating evidence suggests that linear ubiquitin chains are of physiological significance. Dysregulation of linear ubiquitination triggers chronic inflammation and immunodeficiency via downregulation of linear ubiquitin-dependent nuclear factor-kappa B (NF-κB) signaling and by triggering TNF-α-induced cell death, suggesting that linear ubiquitination is a homeostatic regulator of tissue-specific functions. In this review, we focus on our current understating of the molecular and cellular mechanisms by which linear ubiquitin chains control inflammatory environments. Furthermore, we review the role of linear ubiquitination on T cell development, differentiation, and function, thereby providing insight into its direct association with maintaining the immune system.

Differential involvement of LUBAC-mediated linear ubiquitination in intestinal epithelial cells and macrophages during intestinal inflammation.

Disruption of the intestinal epithelial barrier and dysregulation of macrophages are major factors contributing to the pathogenesis of inflammatory bowel diseases (IBDs). Activation of NF-κB and cell death are involved in maintaining intestinal homeostasis in a cell type-dependent manner. Although both are regulated by linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination, the physiological relevance of linear ubiquitination to intestinal inflammation remains unexplored. Here, we used two experimental mouse models of IBD (intraperitoneal LPS and oral dextran sodium sulfate [DSS] administration) to examine the role of linear ubiquitination in intestinal epithelial cells (IECs) and macrophages during intestinal inflammation. We did this by deleting the linear ubiquitination activity of LUBAC specifically from IECs or macrophages. Upon LPS administration, loss of ligase activity in IECs induced mucosal inflammation and augmented IEC death. LPS-mediated death of LUBAC-defective IECs was triggered by TNF. IEC death was rescued by an anti-TNF antibody, and TNF (but not LPS) induced apoptosis of organoids derived from LUBAC-defective IECs. However, augmented TNF-mediated IEC death did not overtly affect the severity of colitis after DSS administration. By contrast, defective LUBAC ligase activity in macrophages ameliorated DSS-induced colitis by attenuating both infiltration of macrophages and expression of inflammatory cytokines. Decreased production of macrophage chemoattractant MCP-1/CCL2, as well as pro-inflammatory IL-6 and TNF, occurred through impaired activation of NF-κB and ERK via loss of ligase activity in macrophages. Taken together, these results indicate that both intraperitoneal LPS and oral DSS administrations are beneficial for evaluating epithelial integrity under inflammatory conditions, as well as macrophage functions in the event of an epithelial barrier breach. The data clarify the cell-specific roles of linear ubiquitination as a critical regulator of TNF-mediated epithelial integrity and macrophage pro-inflammatory responses during intestinal inflammation. © 2022 The Pathological Society of Great Britain and Ireland.

Assembly pathway of the Mammalian proteasome base subcomplex is mediated by multiple specific chaperones.

The 26S proteasome is an enzymatic complex that degrades ubiquitinated proteins in eukaryotic cells. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The latter is further divided into the lid and base subcomplexes. While the mechanism involved in the assembly of the CP is well investigated, that of the RP is poorly understood. Here, we show that the formation of the mammalian base subcomplex involves three distinct modules, where specific pairs of ATPase subunits are associated with the distinct chaperones p28, S5b, or p27. The process of base formation starts from association of the p28-Rpt3-Rpt6-Rpn14 complex with the S5b-Rpt1-Rpt2-Rpn1 complex, followed by incorporation of the p27-Rpt5-Rpt4 complex and Rpn2, where p28, S5b, and p27 regulate the associations between the modules. These chaperones dissociate before completion of 26S proteasome formation. Our results demonstrate that base assembly is facilitated by multiple proteasome-dedicated chaperones, like CP assembly.

LUBAC accelerates B-cell lymphomagenesis by conferring resistance to genotoxic stress on B cells.

The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death and is a suitable therapeutic target for B-cell lymphomas.

Thymoproteasome shapes immunocompetent repertoire of CD8+ T cells.

How self-peptides displayed in the thymus contribute to the development of immunocompetent and self-protective T cells is largely unknown. In contrast, the role of thymic self-peptides in eliminating self-reactive T cells and thereby preventing autoimmunity is well established. A type of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (cTECs) and is required for the generation of optimal cellularity of CD8+ T cells. Here, we show that cTECs displayed thymoproteasome-specific peptide-MHC class I complexes essential for the positive selection of major and diverse repertoire of MHC class I-restricted T cells. CD8+ T cells generated in the absence of thymoproteasomes displayed a markedly altered T cell receptor repertoire that was defective in both allogeneic and antiviral responses. These results demonstrate that thymoproteasome-dependent self-peptide production is required for the development of an immunocompetent repertoire of CD8+ T cells.

Roles of linear ubiquitinylation, a crucial regulator of NF-κB and cell death, in the immune system.

Linear ubiquitinylation, a newly identified post-translational modification, is catalyzed by the linear ubiquitin assembly complex (LUBAC), which is composed of three different subunits, HOIL-1L (heme-oxidized IRP2 ligase 1L), HOIP (HOIL-1 interacting protein), and SHARPIN (SHANK-associated RH domain-interacting protein). LUBAC plays a critical role in the activation of nuclear factor-κB (NF-κB) signaling triggered by a variety of stimuli, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and pathogen-derived components, and in the protection from cell death. Loss of function of SHARPIN in mice triggers chronic inflammation in multiple organs including the skin, as well as immunodeficiency. In humans, mutations in the gene encoding HOIL-1L cause chronic hyperinflammation and immunodeficiency, which are both associated with decreased levels of LUBAC. The linear ubiquitinylation activity of LUBAC is indispensable for B-cell function in mice, and hyperactivation of LUBAC is associated with oncogenesis in certain forms of B-cell lymphoma. In this review, the current understanding of the biochemistry of LUBAC-mediated linear ubiquitinylation and its involvement in the immune system are discussed.

Relapsing-remitting central nervous system autoimmunity mediated by GFAP-specific CD8 T cells.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons, and axons. Historically, MS has been thought to be a CD4 T cell-mediated autoimmune disease of CNS white matter. However, recent studies identified CD8 T cell infiltrates and gray matter lesions in MS patients. These findings suggest that CD8 T cells and CNS Ags other than myelin proteins may be involved during the MS disease process. In this article, we show that CD8 T cells reactive to glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes, can avoid tolerance mechanisms and, depending upon the T cell-triggering event, drive unique aspects of inflammatory CNS autoimmunity. In GFAP-specific CD8 TCR-transgenic (BG1) mice, tissue resident memory-like CD8 T cells spontaneously infiltrate the gray matter and white matter of the CNS, resulting in a relapsing-remitting CNS autoimmunity. The frequency, severity, and remissions from spontaneous disease are controlled by the presence of polyclonal B cells. In contrast, a viral trigger induces GFAP-specific CD8 T effector cells to exclusively target the meninges and vascular/perivascular space of the gray and white matter of the brain, causing a rapid, acute CNS disease. These findings demonstrate that the type of CD8 T cell-triggering event can determine the presentation of distinct CNS autoimmune disease pathologies.

Measurement of Proteasome Activity in Peripheral Blood Mononuclear Cells as an Indicator of Susceptibility to Bortezomib-Induced Severe Neurological Adverse Events in Patients with Multiple Myeloma.

AIMS: To explore the biomarker for predicting the occurrence of adverse events in myeloma patients treated by intravenous bortezomib, we measured proteasome activity in peripheral blood mononuclear cells. METHODS: Samples were obtained from 34 bortezomib-naïve patients. Proteasome activity was measured at pre- and postchemotherapy phase by using a synthetic substrate. RESULTS: Bortezomib injection resulted in a dramatic decrease in proteasome activity, reaching 32.4 ± 18.79% (mean ± SD) of the pretreatment level at 1 h, but it generally recovered at the end of the first course. In total, 6 patients manifested with severe bortezomib-induced peripheral neuropathy (sBIPN) in the second-third course. There was a nonsignificant trend for these patients to have lower levels of the relative proteasome activity at the end of the first course than those without sBIPN (median: 74.03 vs. 103.2%, p = 0.052). Moreover, in all of them, proteasome activity did not recover to the pretreatment level, whereas no patients with complete recovery manifested with sBIPN. Analysis with Fisher's exact test demonstrated that incomplete recovery of proteasome activity is a significant risk factor for sBIPN (p = 0.014). CONCLUSION: Patients with incomplete recovery of proteasome activity are at high risk for developing sBIPN, and the susceptible patients can be indicated by monitoring proteasome activity.

Microstructure characterization of defects in cubic silicon carbide using transmission electron microscopy.

Microstructures of 3C-SiC grown by chemical vapor deposition (CVD) technique on undulant silicon substrate and a further developed technique called switch-back epitaxy (SBE) were studied using transmission electron microscopy (TEM). In case of the CVD sample, the density of the stacking faults was found to be significantly decreasing along growth direction. Sites of collision of stacking faults were observed using high-resolution transmission electron microscopy. Some of the stacking faults were observed to have disappeared after colliding into each other. The stacking faults were identified to be on the same type of plane and had the same type of displacement vector not only in CVD and SBE but also in the epitaxial layer on the SBE SiC samples.

LUBAC-mediated linear ubiquitination in tissue homeostasis and disease.

In addition to its role in the ubiquitin-proteasome system of protein degradation, polyubiquitination is involved in the regulation of intracellular events. Depending on the type of ubiquitin-ubiquitin linkage used, polyubiquitin can assume several types of structures. The spatiotemporal dynamics of polyubiquitin involve multiple adaptor proteins and induce different downstream outputs. Linear ubiquitination, in which the N-terminal methionine on the acceptor ubiquitin serves as the site for ubiquitin-ubiquitin conjugation, is a rare and atypical type of polyubiquitin modification. The production of linear ubiquitin chains is dependent on various external inflammatory stimuli and leads to the transient activation of the downstream NF-κB signalling pathway. This in turn suppresses extrinsic programmed cell death signals and protects cells from activation-induced cell death under inflammatory conditions. Recent evidence has revealed the role of linear ubiquitination in various biological processes under both physiological and pathological conditions. This led us to propose that linear ubiquitination may be pivotal in the 'inflammatory adaptation' of cells, and consequently in tissue homeostasis and inflammatory disease. In this review, we focused on the physiological and pathophysiological roles of linear ubiquitination in vivo in response to a changing inflammatory microenvironment.

Linear ubiquitination-induced necrotic tumor remodeling elicits immune evasion.

Tumor-elicited inflammation confers tumorigenic properties, including cell death resistance, proliferation, or immune evasion. To focus on inflammatory signaling in tumors, we investigated linear ubiquitination, which enhances the nuclear factor-κB signaling pathway and prevents extrinsic programmed cell death under inflammatory environments. Here, we showed that linear ubiquitination was augmented especially in tumor cells around a necrotic core. Linear ubiquitination allowed melanomas to tolerate the hostile tumor microenvironment and to extend a necrosis-containing morphology. Loss of linear ubiquitination resulted in few necrotic lesions and growth regression, further leading to repression of innate anti-PD-1 therapy resistance signatures in melanoma as well as activation of interferon responses and antigen presentation that promote immune-mediated tumor eradication. Collectively, linear ubiquitination promotes tumor-specific tissue remodeling and the ensuing immune evasion.

Genetic deletion and pharmacologic inhibition of E3 ubiquitin ligase HOIP impairs the propagation of myeloid leukemia.

We investigated the role of Hoip, a catalytic subunit of linear ubiquitin chain assembly complex (LUBAC), in adult hematopoiesis and myeloid leukemia by using both conditional deletion of Hoip and small-molecule chemical inhibitors of Hoip. Conditional deletion of Hoip led to significantly longer survival and marked depletion of leukemia burden in murine myeloid leukemia models. Nevertheless, a competitive transplantation assay showed the reduction of donor-derived cells in the bone marrow of recipient mice was relatively mild after conditional deletion of Hoip. Although both Hoip-deficient hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) impaired the maintenance of quiescence, conditional deletion of Hoipinduced apoptosis in LSCs but not HSCs in vivo. Structure-function analysis revealed that LUBAC ligase activity and the interaction of LUBAC subunits were critical for the propagation of leukemia. Hoip regulated oxidative phosphorylation pathway independently of nuclear factor kappa B pathway in leukemia, but not in normal hematopoietic cells. Finally, the administration of thiolutin, which inhibits the catalytic activity of Hoip, improved the survival of recipients in murine myeloid leukemia and suppressed propagation in the patient-derived xenograft model of myeloid leukemia. Collectively, these data indicate that inhibition of LUBAC activity may be a valid therapeutic target for myeloid leukemia.

A method for accurate temperature measurement using infrared thermal camera.

The temperature distribution on a centre-holed thin foil of molybdenum, used as a sample and heated using a sample-heating holder for electron microscopy, was measured using an infrared thermal camera. The temperature on the heated foil area located near the heating stage of the heating holder is almost equal to the temperature on the heating stage. However, during the measurement of the temperature at the edge of the hole of the foil located farthest from the heating stage, a drop in temperature should be taken into consideration; however, so far, no method has been developed to locally measure the temperature distribution on the heated sample. In this study, a method for the accurate measurement of temperature distribution on heated samples for electron microscopy is discussed.

ABIN1 is a signal-induced autophagy receptor that attenuates NF-κB activation by recognizing linear ubiquitin chains.

Linear ubiquitin chains play pivotal roles in immune signaling by augmenting NF-κB activation and suppressing programmed cell death induced by various stimuli. A20-binding inhibitor of NF-κB 1 (ABIN1) binds to linear ubiquitin chains and attenuates NF-κB activation and cell death induction. Although interactions with linear ubiquitin chains are thought to play a role in ABIN1-mediated suppression of NF-κB and cell death, the underlying molecular mechanisms remain unclear. Here, we show that upon stimulation by Toll-like receptor (TLR) ligands, ABIN1 is phosphorylated on Ser 83 and functions as a selective autophagy receptor. ABIN1 recognizes components of the MyD88 signaling complex via interaction with linear ubiquitin chains conjugated to components of the complex in TLR signaling, which leads to autophagic degradation of signaling proteins and attenuated NF-κB signaling. Our current findings indicate that phosphorylation and linear ubiquitination also play a role in downregulation of signaling via selective induction of autophagy.

Rpn10-mediated degradation of ubiquitinated proteins is essential for mouse development.

Rpn10 is a subunit of the 26S proteasome that recognizes polyubiquitinated proteins. The importance of Rpn10 in ubiquitin-mediated proteolysis is debatable, since a deficiency of Rpn10 causes different phenotypes in different organisms. To date, the role of mammalian Rpn10 has not been examined genetically. Moreover, vertebrates have five splice variants of Rpn10 whose expressions are developmentally regulated, but their biological significance is not understood. To address these issues, we generated three kinds of Rpn10 mutant mice. Rpn10 knockout resulted in early-embryonic lethality, demonstrating the essential role of Rpn10 in mouse development. Rpn10a knock-in mice, which exclusively expressed the constitutive type of Rpn10 and did not express vertebrate-specific variants, grew normally, indicating that Rpn10 diversity is not essential for conventional development. Mice expressing the N-terminal portion of Rpn10, which contained a von Willebrand factor A (VWA) domain but lacked ubiquitin-interacting motifs (Rpn10DeltaUIM), also exhibited embryonic lethality, suggesting the important contribution of UIM domains to viability, but survived longer than Rpn10-null mice, consistent with a "facilitator" function of the VWA domain. Biochemical analysis of the Rpn10DeltaUIM liver showed specific impairment of degradation of ubiquitinated proteins. Our results demonstrate that Rpn10-mediated degradation of ubiquitinated proteins, catalyzed by UIMs, is indispensable for mammalian life.

In-situ TEM studies of the sintering behavior of copper nanoparticles covered by biopolymer nanoskin.

Sintering behavior of copper nanoparticles with a protective layer of gelatin synthesized by wet-chemical process with an average diameter of 45 nm has been observed using in-situ transmission electron microscopy (TEM). Copper nanoparticles were sublimated without sintering at about 925 degrees C at 2.0 x 10(-)(5) Pa, and carbonized gelatin remained and retained the shape of the initial layer of nanoparticles. Copper nanoparticles were sintered without sublimation at about 250 degrees C with between 1.0 x 10(-)(4) and 6.0 x 10(-)(4) Pa of oxygen gas flow. It was found that the surface of the sintered copper was covered by a gelatin layer.

Measurement of electric field distribution using a conventional transmission electron microscope.

A simple and quick method to measure the electric field distribution near a specimen using a conventional transmission electron microscope has been developed. The electric field distribution around a field emitter needle was measured to evaluate the performance of the method. It was found that this method allows us to measure the 2D distribution of electric field quantitatively with error of <10% with submicron spatial resolution. The dedicated aperture and software to construct an automated analysis system have been developed.

The HOIL-1L ligase modulates immune signalling and cell death via monoubiquitination of LUBAC.

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.

Modulation of autoimmune pathogenesis by T cell-triggered inflammatory cell death.

T cell-mediated autoimmunity encompasses diverse immunopathological outcomes; however, the mechanisms underlying this diversity are largely unknown. Dysfunction of the tripartite linear ubiquitin chain assembly complex (LUBAC) is associated with distinct autonomous immune-related diseases. Cpdm mice lacking Sharpin, an accessory subunit of LUBAC, have innate immune cell-predominant dermatitis triggered by death of LUBAC-compromised keratinocytes. Here we show that specific gene ablation of Sharpin in mouse Treg causes phenotypes mimicking cpdm-like inflammation. Mechanistic analyses find that multiple types of programmed cell death triggered by TNF from tissue-oriented T cells initiate proinflammatory responses to implicate innate immune-mediated pathogenesis in this T cell-mediated inflammation. Moreover, additional disruption of the Hoip locus encoding the catalytic subunit of LUBAC converts cpdm-like dermatitis to T cell-predominant autoimmune lesions; however, innate immune-mediated pathogenesis still remains. These findings show that T cell-mediated killing and sequential autoinflammation are common and crucial for pathogenic diversity during T cell-mediated autoimmune responses.