In the moonlight: non-catalytic functions of ubiquitin and ubiquitin-like proteases.

Proteases that cleave ubiquitin or ubiquitin-like proteins (UBLs) are critical players in maintaining the homeostasis of the organism. Concordantly, their dysregulation has been directly linked to various diseases, including cancer, neurodegeneration, developmental aberrations, cardiac disorders and inflammation. Given their potential as novel therapeutic targets, it is essential to fully understand their mechanisms of action. Traditionally, observed effects resulting from deficiencies in deubiquitinases (DUBs) and UBL proteases have often been attributed to the misregulation of substrate modification by ubiquitin or UBLs. Therefore, much research has focused on understanding the catalytic activities of these proteins. However, this view has overlooked the possibility that DUBs and UBL proteases might also have significant non-catalytic functions, which are more prevalent than previously believed and urgently require further investigation. Moreover, multiple examples have shown that either selective loss of only the protease activity or complete absence of these proteins can have different functional and physiological consequences. Furthermore, DUBs and UBL proteases have been shown to often contain domains or binding motifs that not only modulate their catalytic activity but can also mediate entirely different functions. This review aims to shed light on the non-catalytic, moonlighting functions of DUBs and UBL proteases, which extend beyond the hydrolysis of ubiquitin and UBL chains and are just beginning to emerge.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here we show that its lysosomal degradation is dependent on ubiquitylation at Lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4 and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells and in cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype, but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive, nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4, or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

Compounds derived from Humulus lupulus inhibit SARS-CoV-2 papain-like protease and virus replication.

BACKGROUND: Selected natural compounds exhibit very good antiviral properties. Especially, the medicinal plant Humulus lupulus (hop) contains several secondary plant metabolites some of which have previously shown antiviral activities. Among them, the prenylated chalcone xanthohumol (XN) demonstrated to be a potent inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). HYPOTHESIS/PURPOSE: Following the finding that xanthohumol (XN) is a potent inhibitor of SARS-CoV-2 Mpro, the effect of XN and its major derivatives isoxanthohumol (IXN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) from hops on SARS-CoV-2 papain-like protease (PLpro) were investigated. STUDY DESIGN: The modulatory effect of the hop compounds on PLpro were studied first in silico and then in vitro. In addition, the actual effect of hop compounds on the replication of SARS-CoV-2 in host cells was investigated. METHODS: In silico docking analysis was used to predict the binding affinity of hop compounds to the active site of PLpro. A recombinant PLpro was cloned, purified, characterized, and analyzed by small-angle X-ray scattering (SAXS), deISGylation assays, and kinetic analyses. Antiviral activity of hop compounds was assessed using the fluorescently labeled wildtype SARS-CoV-2 (icSARS-CoV-2-mNG) in Caco-2 host cells. RESULTS: Our in silico docking suggests that the purified hop compounds bind to the active site of SARS-CoV-2 PLpro blocking the access of its natural substrates. The hop-derived compounds inhibit SARS-CoV-2 PLpro with half maximal inhibitory concentration (IC50) values in the range of 59-162 µM. Furthermore, we demonstrate that XN and 6-PN, in particular, impede viral replication with IC50 values of 3.3 µM and 7.3 µM, respectively. CONCLUSION: In addition to the already known inhibition of Mpro by XN, our results show, for the first time, that hop-derived compounds target also SARS-CoV-2 PLpro which is a promising therapeutic target as it contributes to both viral replication and modulation of the immune system. These findings support the possibility to develop new hop-derived antiviral drugs targeting human coronaviruses.

Downregulation of Ubiquitin-Specific Protease 15 (USP15) Does Not Provide Therapeutic Benefit in Experimental Mesial Temporal Lobe Epilepsy.

Structural epilepsies display complex immune activation signatures. However, it is unclear which neuroinflammatory pathways drive pathobiology. Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor ß, interferon α/ß, and nuclear factor erythroid 2-related factor 2 pathways. Since these pathways are regulated by ubiquitin-specific proteases (USP), in particular USP15, we hypothesized that USP15 blockade may provide therapeutic relief in treatment-resistant epilepsies. For validation, transgenic mice which either constitutively or inducibly lack Usp15 gene expression underwent intrahippocampal kainate injections to induce mTLE. We show that the severity of status epilepticus is unaltered in mice constitutively lacking Usp15 compared to wild types. Cell death, reactive gliosis, and changes in the inflammatory transcriptome were pronounced at 4 days after kainate injection. However, these brain inflammation signatures did not differ between genotypes. Likewise, induced deletion of Usp15 in chronic epilepsy did not affect seizure generation, cell death, gliosis, or the transcriptome. Concordantly, siRNA-mediated knockdown of Usp15 in a microglial cell line did not impact inflammatory responses in the form of cytokine release. Our data show that a lack of USP15 is insufficient to modulate the expression of relevant neuroinflammatory pathways in an mTLE mouse model and do not support targeting USP15 as a therapeutic approach for pharmacoresistant epilepsy.

USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

A comparative analysis of microglial inducible Cre lines.

Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has particularly benefited because microglia historically have been difficult to transduce with virus or electroporation methods for gene delivery. Here, we investigate five of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency, cell-type specificity, and spontaneous recombination, depending on the Cre line and inter-loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency, particularly in microglia. There is increasing evidence that microglia are key regulators of neural circuits and major drivers of a broad range of neurological diseases. Reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field and the development of microglia-based therapeutics.

A comparative analysis of microglial inducible Cre lines.

Cre/LoxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. The field of microglial biology has particularly benefited from this technology as microglia have historically been difficult to transduce with virus or electroporation methods for gene delivery. Here, we interrogate four of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency and spontaneous recombination, depending on the Cre line and loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency in microglia, which could be extended to other cell types. There is increasing evidence that microglia are key regulators of neural circuit structure and function. Microglia are also major drivers of a broad range of neurological diseases. Thus, reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field of microglial biology and the development of microglia-based therapeutics.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity.

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

Deubiquitinating Enzyme USP8 Is Essential for Skeletogenesis by Regulating Wnt Signaling.

Disturbance in a differentiation program of skeletal stem cells leads to indecorous skeletogenesis. Growing evidence suggests that a fine-tuning of ubiquitin-mediated protein degradation is crucial for skeletal stem cells to maintain their stemness and osteogenic potential. Here, we demonstrate that the deubiquitinating enzyme (DUB) ubiquitin-specific protease 8 (USP8) stabilizes the Wnt receptor frizzled 5 (FZD5) by preventing its lysosomal degradation. This pathway is essential for Wnt/ß-catenin signaling and the differentiation of osteoprogenitors to mature osteoblasts. Accordingly, deletion of USP8 in osteoprogenitors (Usp8Osx) resulted in a near-complete blockade in skeletal mineralization, similar to that seen in mice with defective Wnt/ß-catenin signaling. Likewise, transplanting USP8-deficient osteoprogenitors under the renal capsule in wild-type secondary hosts did not to induce bone formation. Collectively, this study unveils an essential role for the DUB USP8 in Wnt/ß-catenin signaling in osteoprogenitors and osteogenesis during skeletal development.

SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis.

Microtubule depolymerases of the kinesin-13 family play important roles in various cellular processes and are frequently overexpressed in different cancer types. Despite the importance of their correct abundance, remarkably little is known about how their levels are regulated in cells. Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5 , including the kinesin-13 member Kif2c/MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2a and Kif2b become efficiently polyubiquitylated by neddylated SCFFbxw5 and Cdc34, without requiring preceding modifications. In cells, SCFFbxw5  targets MCAK for proteasomal degradation predominantly during G2 . While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies and impairs ciliogenesis in the following G1 /G0 , which can be rescued by concomitant knockdown of MCAK, Kif2a or Kif2b. We thus propose a novel regulatory event of ciliogenesis that begins already within the G2 phase of the preceding cell cycle.

Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions.

COVID-19 can cause severe neurological symptoms, but the underlying pathophysiological mechanisms are unclear. Here, we interrogated the brain stems and olfactory bulbs in postmortem patients who had COVID-19 using imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional, single-cell level and compared their immune map to non-COVID respiratory failure, multiple sclerosis, and control patients. We observed substantial immune activation in the central nervous system with pronounced neuropathology (astrocytosis, axonal damage, and blood-brain-barrier leakage) and detected viral antigen in ACE2-receptor-positive cells enriched in the vascular compartment. Microglial nodules and the perivascular compartment represented COVID-19-specific, microanatomic-immune niches with context-specific cellular interactions enriched for activated CD8+ T cells. Altered brain T-cell-microglial interactions were linked to clinical measures of systemic inflammation and disturbed hemostasis. This study identifies profound neuroinflammation with activation of innate and adaptive immune cells as correlates of COVID-19 neuropathology, with implications for potential therapeutic strategies.

Evidence for an involvement of the ubiquitin-like modifier ISG15 in MHC class I antigen presentation.

The IFN stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I IFNs and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by IFN-ß. The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to WT mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing.

Author Correction: Novel Hexb-based tools for studying microglia in the CNS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

Interferon-stimulated gene 15 accelerates replication fork progression inducing chromosomal breakage.

DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-ß, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.

Novel Hexb-based tools for studying microglia in the CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming.

Protein modification with ISG15 (ISGylation) represents a major type I IFN-induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies.