MST1 Negatively Regulates TNFα-Induced NF-κB Signaling through Modulating LUBAC Activity.

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

Mechanistic insights into the subversion of the linear ubiquitin chain assembly complex by the E3 ligase IpaH1.4 of Shigella flexneri.

Shigella flexneri, a gram-negative bacterium, is the major culprit of bacterial shigellosis and causes a large number of human infection cases and deaths worldwide annually. For evading the host immune response during infection, S. flexneri secrets two highly similar E3 ligases, IpaH1.4 and IpaH2.5, to subvert the linear ubiquitin chain assembly complex (LUBAC) of host cells, which is composed of HOIP, HOIL-1L, and SHARPIN. However, the detailed molecular mechanism underpinning the subversion of the LUBAC by IpaH1.4/2.5 remains elusive. Here, we demonstrated that IpaH1.4 can specifically recognize HOIP and HOIL-1L through its leucine-rich repeat (LRR) domain by binding to the HOIP RING1 domain and HOIL-1L ubiquitin-like (UBL) domain, respectively. The determined crystal structures of IpaH1.4 LRR/HOIP RING1, IpaH1.4 LRR/HOIL-1L UBL, and HOIP RING1/UBE2L3 complexes not only elucidate the binding mechanisms of IpaH1.4 with HOIP and HOIL-1L but also unveil that the recognition of HOIP by IpaH1.4 can inhibit the E2 binding of HOIP. Furthermore, we demonstrated that the interaction of IpaH1.4 LRR with HOIP RING1 or HOIL-1L UBL is essential for the ubiquitination of HOIP or HOIL-1L in vitro as well as the suppression of NF-κB activation by IpaH1.4 in cells. In summary, our work elucidated that in addition to inducing the proteasomal degradation of LUBAC, IpaH1.4 can also inhibit the E3 activity of LUBAC by blocking its E2 loading and/or disturbing its stability, thereby providing a paradigm showing how a bacterial E3 ligase adopts multiple tactics to subvert the key LUBAC of host cells.

Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated HoipK778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, HoipK778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in HoipK778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.

AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex in which the ubiquitin-like (UBL) domains of SHARPIN and HOIL-1L interact with HOIP to determine the structural stability of LUBAC. The interactions between subunits within LUBAC have been a topic of extensive research. However, the impact of the LTM motif on the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP remains unclear. Here, we discover that the absence of the LTM motif in the AlphaFold2-predicted LUBAC structure alters the HOIP-UBA structure. We employ GeoPPI to calculate the changes in binding free energy (ΔG) caused by single-point mutations between subunits, simulating their protein-protein interactions. The results reveal that the presence of the LTM motif decreases the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP, leading to a decrease in the structural stability of LUBAC. Furthermore, using the AlphaFold2-predicted results, we find that HOIP (629‒695) and HOIP-UBA bind to both sides of HOIL-1L-UBL, respectively. The experiments of Gromacs molecular dynamics simulations, SPR and ITC demonstrate that the elongated domain formed by HOIP (629‒695) and HOIP-UBA, hereafter referred to as the HOIP (466‒695) structure, interacts with HOIL-1L-UBL to form a structurally stable complex. These findings illustrate the collaborative interaction between HOIP-UBA and HOIP (629‒695) with HOIL-1L-UBL, which influences the structural stability of LUBAC.

HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma-induced apoptosis.

The success of cancer immunotherapy is limited to a subset of patients, highlighting the need to identify the processes by which tumors evade immunity. Using CRISPR/Cas9 screening, we reveal that melanoma cells lacking HOIP, the catalytic subunit of LUBAC, are highly susceptible to both NK and CD8+ T-cell-mediated killing. We demonstrate that HOIP-deficient tumor cells exhibit increased sensitivity to the combined effect of the inflammatory cytokines, TNF and IFN-γ, released by NK and CD8+ T cells upon target recognition. Both genetic deletion and pharmacological inhibition of HOIP augment tumor cell sensitivity to combined TNF and IFN-γ. Together, we unveil a protective regulatory axis, involving HOIP, which limits a transcription-dependent form of cell death that engages both intrinsic and extrinsic apoptotic machinery upon exposure to TNF and IFN-γ. Our findings highlight HOIP inhibition as a potential strategy to harness and enhance the killing capacity of TNF and IFN-γ during immunotherapy.

LUBAC: a new player in polyglucosan body disease.

Altered protein ubiquitination is associated with the pathobiology of numerous diseases; however, its involvement in glycogen metabolism and associated polyglucosan body (PB) disease has not been investigated in depth. In PB disease, excessively long and less branched glycogen chains (polyglucosan bodies, PBs) are formed, which precipitate in different tissues causing myopathy, cardiomyopathy and/or neurodegeneration. Linear ubiquitin chain assembly complex (LUBAC) is a multi-protein complex composed of two E3 ubiquitin ligases HOIL-1L and HOIP and an adaptor protein SHARPIN. Together they are responsible for M1-linked ubiquitination of substrates primarily related to immune signaling and cell death pathways. Consequently, severe immunodeficiency is a hallmark of many LUBAC deficient patients. Remarkably, all HOIL-1L deficient patients exhibit accumulation of PBs in different organs especially skeletal and cardiac muscle resulting in myopathy and cardiomyopathy with heart failure. This emphasizes LUBAC's important role in glycogen metabolism. To date, neither a glycogen metabolism-related LUBAC substrate nor the molecular mechanism are known. Hence, current reviews on LUBAC's involvement in glycogen metabolism are lacking. Here, we aim to fill this gap by describing LUBAC's involvement in PB disease. We present a comprehensive review of LUBAC structure, its role in M1-linked and other types of atypical ubiquitination, PB pathology in human patients and findings in new mouse models to study the disease. We conclude the review with recent drug developments and near-future gene-based therapeutic approaches to treat LUBAC related PB disease.

Linear Ubiquitination Mediates EGFR-Induced NF-κB Pathway and Tumor Development.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR and that perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.

SPATA2 promotes CYLD activity and regulates TNF-induced NF-κB signaling and cell death.

K63- and Met1-linked ubiquitylation are crucial posttranslational modifications for TNF receptor signaling. These non-degradative ubiquitylations are counteracted by deubiquitinases (DUBs), such as the enzyme CYLD, resulting in an appropriate signal strength, but the regulation of this process remains incompletely understood. Here, we describe an interaction partner of CYLD, SPATA2, which we identified by a mass spectrometry screen. We find that SPATA2 interacts via its PUB domain with CYLD, while a PUB interaction motif (PIM) of SPATA2 interacts with the PUB domain of the LUBAC component HOIP SPATA2 is required for the recruitment of CYLD to the TNF receptor signaling complex upon TNFR stimulation. Moreover, SPATA2 acts as an allosteric activator for the K63- and M1-deubiquitinase activity of CYLD In consequence, SPATA2 substantially attenuates TNF-induced NF-κB and MAPK signaling. Conversely, SPATA2 is required for TNF-induced complex II formation, caspase activation, and apoptosis. Thus, this study identifies SPATA2 as an important factor in the TNF signaling pathway with a substantial role for the effects mediated by the cytokine.

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.

RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.

Charge Transport between Coupling Colloidal Perovskite Quantum Dots Assisted by Functional Conjugated Ligands.

Long alkyl-chain capping ligands are indispensable for preparing stable colloidal quantum dots. However, its insulating feature blocks efficient carrier transport among QDs, leading to inferior performance in light-emitting diodes (LEDs). The trade-off between conductivity and colloidal stability of QDs has now been overcome. Methylamine lead bromide (MAPbBr3 ) QDs with a conjugated alkyl-amine, 3-phenyl-2-propen-1-amine (PPA), as ligands were prepared. Owing to electron cloud overlapping and the delocalization effect of conjugated molecules, the conductivity and carrier mobility of PPA-QDs films increased almost 22 times over that of OA-QD films without compromising colloidal stability and photoluminescence. PPA-QDs LEDs exhibit a maximum current efficiency of 9.08 cd A-1 , which is 8 times of that of OA-QDs LEDs (1.14 cd A-1 ). This work provides critical solution for the poor conductivity of QDs in applications of energy-related devices.

A novel HOIP frameshift variant alleviates NF-kappaB signalling and sensitizes cells to TNF-induced death.

BACKGROUND: HOIP is the catalytic subunit of the E3 ligase complex (linear ubiquitin chain assembly complex), which is able to generate linear ubiquitin chains. However, the role of rare HOIP functionally deficient variants remains unclear. The pathogenic mechanism and the relationship with immune deficiency phenotypes remain to be clarified. METHODS: Based on a next-generation sequencing panel of 270 genes, we identified a HOIP deletion variant that causes common variable immunodeficiency disease. Bioinformatics analysis and cell-based experiments were performed to study the molecular mechanism by which the variant causes immunodeficiency diseases. FINDINGS: A homozygous loss-of-function variant in HOIP was identified. The variant causes a frameshift and generates a premature termination codon in messenger RNA, resulting in a C-terminal truncated HOIP mutant, that is, the loss of the linear ubiquitin chain-specific catalytic domain. The truncated HOIP mutant has impaired E3 ligase function in linear ubiquitination, leading to the suppression of canonical NF-κB signalling and increased TNF-induced multiple forms of cell death. INTERPRETATION: The loss-of-function HOIP variant accounts for the immune deficiencies. The canonical NF-κB pathway and cell death are involved in the pathogenesis of the disease. FUNDING: This study was funded by the National Natural Science Foundation of China (No. 82270444 and 81501851). RESEARCH IN CONTEXT: Evidence before this study LUBAC is the only known linear ubiquitin chain assembly complex for which HOIP is an essential catalytic subunit. Three HOIP variants have now been identified in two immunodeficient patients and functionally characterised. However, there have been no reports on the pathogenicity of only catalytic domain deletion variants in humans, or the pathogenic mechanisms of catalytic domain deletion variants. Added value of this study We report the first case of an autosomal recessive homozygous deletion variant that results in deletion of the HOIP catalytic structural domain. We demonstrate that this variant is a loss-of-function variant using a heterologous expression system. The variant has impaired E3 ligase function. It can still bind to other subunits of LUBAC, but it fails to generate linear ubiquitin chains. We also explored the underlying mechanisms by which this variant leads to immunodeficiency. The variant attenuates the canonical NF-κB and MAPK signalling cascades and increases the sensitivity of TNFα-induced diverse cell death and activation of mitochondrial apoptosis pathways. These findings provide support for the treatment and drug development of patients with inborn errors of immunity in HOIP and related signalling pathways. Implications of all the available evidence First, this study expands the HOIP pathogenic variant database and phenotypic spectrum. Furthermore, studies on the biological functions of pathogenic variants in relation to the NF-κB signalling pathway and cell death provided new understanding into the genetic basis and pathogenesis of HOIP-deficient immune disease, indicating the necessity of HOIP and related signalling pathway variants as diagnostic targets in patients with similar genetic deficiency phenotypes..

Role of Surface Features on the Initial Dissolution of CH3NH3PbI3 Perovskite in Liquid Water: An Ab Initio Molecular Dynamics Study.

The degradation of CH3NH3PbI3 (MAPbI3) hybrid organic inorganic perovskite (HOIP) by water has been the major issue hampering its use in commercial perovskites solar cells (PSCs), as MAPbI3 HOIP has been known to easily degrade in the presence of water. Even though there have been numerous studies investigating this phenomenon, there is still no consensus on the mechanisms of the initial stages of dissolution. Here, we attempt to consolidate differing mechanistic interpretations previously reported in the literature through the use of the first-principles constrained ab initio molecular dynamics (AIMD) to study both the energetics and mechanisms that accompany the degradation of MAPbI3 HOIP in liquid water. By comparing the dissolution free energy barrier between surface species of different surficial types, we find that the dominant dissolution mechanisms of surface species vary widely based on the specific surface features. The high sensitivity of the dissolution mechanism to surface features has contributed to the many dissolution mechanisms proposed in the literature. In contrast, the dissolution free energy barriers are mainly determined by the dissolving species rather than the type of surfaces, and the type of surfaces the ions are dissolving from is inconsequential toward the dissolution free energy barrier. However, the presence of surface defects such as vacancy sites is found to significantly lower the dissolution free energy barriers. Based on the estimated dissolution free energy barriers, we propose that the dissolution of MAPbI3 HOIP in liquid water originates from surface defect sites that propagate laterally along the surface layer of the MAPbI3 HOIP crystal.

Structural basis for ubiquitylation by HOIL-1.

The linear ubiquitin chain assembly complex synthesises linear Ub chains which constitute a binding and activation platform for components of the TNF signalling pathway. One of the components of LUBAC is the ubiquitin ligase HOIL-1 which has been shown to generate oxyester linkages on several proteins and on linear polysaccharides. We show that HOIL-1 activity requires linear tetra-Ub binding which enables HOIL-1 to mono-ubiquitylate linear Ub chains and polysaccharides. Furthermore, we describe the crystal structure of a C-terminal tandem domain construct of HOIL-1 comprising the IBR and RING2 domains. Interestingly, the structure reveals a unique bi-nuclear Zn-cluster which substitutes the second zinc finger of the canonical RING2 fold. We identify the C-terminal histidine of this bi-nuclear Zn-cluster as the catalytic base required for the ubiquitylation activity of HOIL-1. Our study suggests that the unique zinc-coordinating architecture of RING2 provides a binding platform for ubiquitylation targets.

The E3 Ubiquitin Ligase HOIP inhibits Cancer Cell Apoptosis via modulating PTEN stability.

Chemotherapy is widely used in a variety of solid tumors, such as lung cancer, gastric cancer and breast cancer. The genotoxic drugs, such as cisplatin, suppress cancer progression either by inhibition cell proliferation or facilitating apoptosis. However, the chemotherapy resistance remains an urgent challenge in cancer therapy, especially in advanced stages. Several studies showed that the activation of pro-survival pathways, such as PI3K-AKT, participated in mediating chemotherapy resistance. The insights into the molecular mechanisms for underlying chemotherapy resistance are of great importance to improve cancer patient survival in advanced stages. The HOIP protein belongs to the RING family E3 ubiquitin ligases and modulates several atypical ubiquitination processes in cellular signaling. Previous studies showed that HOIP might be an important effector in modulating cancer cell death under genotoxic drugs. Here, we report that HOIP associates with PTEN and facilitates PTEN degradation in cancer cells. Depletion of HOIP causes cell cycle arrest and apoptosis, which effects could be rescued by PTEN silencing. Besides, the survival data from public available database show that HOIP expression correlates with poor survival in several types of chemotherapy-treated cancer patients. In conclusion, our study establishes a novel mechanism by which HOIP modulates PTEN stability and facilitates chemotherapy resistance in malignancies.

Biochemistry, Pathophysiology, and Regulation of Linear Ubiquitination: Intricate Regulation by Coordinated Functions of the Associated Ligase and Deubiquitinase.

The ubiquitin system modulates protein functions by decorating target proteins with ubiquitin chains in most cases. Several types of ubiquitin chains exist, and chain type determines the mode of regulation of conjugated proteins. LUBAC is a ubiquitin ligase complex that specifically generates N-terminally Met1-linked linear ubiquitin chains. Although linear ubiquitin chains are much less abundant than other types of ubiquitin chains, they play pivotal roles in cell survival, proliferation, the immune response, and elimination of bacteria by selective autophagy. Because linear ubiquitin chains regulate inflammatory responses by controlling the proinflammatory transcription factor NF-κB and programmed cell death (including apoptosis and necroptosis), abnormal generation of linear chains can result in pathogenesis. LUBAC consists of HOIP, HOIL-1L, and SHARPIN; HOIP is the catalytic center for linear ubiquitination. LUBAC is unique in that it contains two different ubiquitin ligases, HOIP and HOIL-1L, in the same ligase complex. Furthermore, LUBAC constitutively interacts with the deubiquitinating enzymes (DUBs) OTULIN and CYLD, which cleave linear ubiquitin chains generated by LUBAC. In this review, we summarize the current status of linear ubiquitination research, and we discuss the intricate regulation of LUBAC-mediated linear ubiquitination by coordinate function of the HOIP and HOIL-1L ligases and OTULIN. Furthermore, we discuss therapeutic approaches to targeting LUBAC-mediated linear ubiquitin chains.

A two-dimensional organic-inorganic lead iodide perovskite: poly[bis(3-fluorocyclobutylammonium) [di-µ-iodido-diiodidoplumbate(VI)]].

In recent years, great technological advances have been achieved in the growth of hybrid organic-inorganic perovskites (HOIPs) and these have attracted extensive attention due to their optoelectronic properties, structural tunability and stability. We present here a new two-dimensional hybrid organic-inorganic perovskite, namely, poly[bis(3-fluorocyclobutylammonium) [di-µ-iodido-diiodidoplumbate(VI)]], {(C4H9FN)2[PbI4]}n, showing a two-dimensional reticular layer with the organic cations in the middle of the meshes. The calculated experimental band gap is 2.44 eV and the band gap is calculated as 2.20 eV theoretically, which further suggests the potential of this compound as a semiconductor.

Correlation of Spatiotemporal Dynamics of Polarization and Charge Transport in Blended Hybrid Organic-Inorganic Perovskites on Macro- and Nanoscales.

Progress in flexible organic electronics necessitates a full understanding of how local inhomogeneities impact electronic and ionic conduction pathways and underlie macroscopic device characteristics. We used frequency- and time-resolved macro- and nanoprobe measurements to study spatiotemporal characteristics of multiscale charge transport dynamics in a series of ternary-blended hybrid organic inorganic perovskites (HOIPs) (MA0.95-xFAxCs0.05PbI3). We show that A-site cation composition defines charge transport mechanisms across broad temporal (102-10-6 s) and spatial (millimeters-picometers) scales. Ab initio molecular dynamic simulations suggest that insertion of FA results in a dynamic lattice, improved ion transport, and dipole screening. We demonstrate that correlations between macro- and nanoscale measurements provide a pathway for accessing distribution of relaxation in nanoscale polarization and charge transport dynamics of ionically conductive functional perovskites.

Second Case of HOIP Deficiency Expands Clinical Features and Defines Inflammatory Transcriptome Regulated by LUBAC.

Background: HOIP is the catalytic subunit of the linear ubiquitination chain assembly complex (LUBAC) that is essential for NF-κB signaling and thus proper innate and adaptive immunity. To date only one patient with HOIP deficiency has been reported with clinical characteristics that include autoinflammation, immunodeficiency, amylopectinosis, and systemic lymphangiectasia. Case: We sought to identify a genetic cause of a disease for an 8 year-old girl who presented with early-onset immune deficiency and autoinflammation. Methods: Targeted next generation sequencing of 352 immune-related genes was performed. Functional studies included transcriptome analysis, cytokine profiling, and protein analysis in patients' primary cells. Results: We identified biallelic variants in close proximity to splice sites (c.1197G>C and c.1737+3A>G) in the RNF31 gene. RNA extracted from patient cells showed alternatively spliced transcripts not present in control cells. Protein expression of HOIP and LUBAC was reduced in primary cells as shown by western blotting. Patient-derived fibroblasts demonstrated attenuated IL-6 production, while PBMCs showed higher TNF production after stimulation with proinflammatory cytokines. RNA sequencing of whole blood RNA and PBMCs demonstrated a marked transcriptome wide change including differential expression of type I interferon regulated genes. Conclusion: We report the second case of HOIP deficiency with novel compound heterozygous mutations in RNF31 and distinct clinical and molecular features. Our results expand on the clinical spectrum of HOIP deficiency and molecular signatures associated with LUBAC deficiency.

Linear Ubiquitin Chains: Cellular Functions and Strategies for Detection and Quantification.

Ubiquitination of proteins is a sophisticated post-translational modification implicated in the regulation of an ever-growing abundance of cellular processes. Recent insights into different layers of complexity have shaped the concept of the ubiquitin code. Key players in determining this code are the number of ubiquitin moieties attached to a substrate, the architecture of polyubiquitin chains, and post-translational modifications of ubiquitin itself. Ubiquitination can induce conformational changes of substrates and alter their interactive profile, resulting in the formation of signaling complexes. Here we focus on a distinct type of ubiquitination that is characterized by an inter-ubiquitin linkage through the N-terminal methionine, called M1-linked or linear ubiquitination. Formation, recognition, and disassembly of linear ubiquitin chains are highly specific processes that are implicated in immune signaling, cell death regulation and protein quality control. Consistent with their role in influencing signaling events, linear ubiquitin chains are formed in a transient and spatially regulated manner, making their detection and quantification challenging.