The missing link? LGMN connects hypoxia and immunosuppression in glioblastoma.

In this issue, Pang and colleagues1 identify the protease legumain as a potential immunotherapy target in glioblastoma that drives tumor-associated macrophages in response to hypoxia.

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control.

OTUB1 is one of the most highly expressed deubiquitinases, counter-regulating the two most abundant ubiquitin chain types. OTUB1 expression is linked to the development and progression of lung cancer and idiopathic pulmonary fibrosis in humans. However, the physiological function of OTUB1 is unknown. Here, we show that constitutive whole-body Otub1 deletion in mice leads to perinatal lethality by asphyxiation. Analysis of (single-cell) RNA sequencing and proteome data demonstrated that OTUB1 is expressed in all lung cell types with a particularly high expression during late-stage lung development (E16.5, E18.5). At E18.5, the lungs of animals with Otub1 deletion presented with increased cell proliferation that decreased saccular air space and prevented inhalation. Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types. Adult mice with conditional whole-body Otub1 deletion (wbOtub1del/del ) also displayed increased lung cell proliferation in addition to hyperventilation and failure to adapt the respiratory pattern to hypoxia. On the molecular level, Otub1 deletion enhanced mTOR signaling in embryonic and adult lung tissues. Based on these results, we propose that OTUB1 is a negative regulator of mTOR signaling with essential functions for lung cell proliferation, lung development, adult lung tissue homeostasis, and respiratory regulation.

Legumain knockout improved cognitive impairment via reducing neuroinflammation in right unilateral common carotid artery occlusion mice.

AIMS: Chronic cerebral hypoperfusion (CCH) is a state of chronic cerebral blood flow reduction, and it is the main cause of cognitive impairment and neurodegenerative diseases. The abnormal upregulation of legumain, a lysosomal cysteine protease, trigger synaptic plasticity impairment and neuroinflammation, which are involved in the underlying pathophysiology of CCH. At present, few studies have reported the role of legumain in cognitive impairment caused by CCH. In our study, we aimed to investigate the involvement of legumain knockout in cognitive function and neuroinflammation in a CCH mouse model. MAIN METHODS: In this study, right unilateral common carotid artery occlusion (rUCCAO) was used to simulate the pathological state of cerebral ischemic injury. Various behavioural tests were executed to assess cognitive performance. In vivo electrophysiological recordings were used to measure synaptic functions. Western blotting, Golgi staining, haematoxylin/eosin staining, and immunofluorescence assays were conducted to examine pathological changes and molecular mechanisms. KEY FINDINGS: The data showed that the level of legumain was significantly increased in the hippocampus of mice subjected to rUCCAO. Legumain knockout significantly improved cognitive function and synaptic plasticity induced by rUCCAO, suggesting that legumain knockout-regulation effectively protected against CCH-induced behavioural dysfunctions. Moreover, legumain knockout suppressed rUCCAO-induced microglial activation, reduced the abnormal expression of inflammatory cytokines and the inflammasome complex, and impeded the activation of P65 and pyroptosis. SIGNIFICANCE: These findings suggest that legumain is an effective regulator of CCH, and may be an ideal target for the development of cerebral ischemia treatments in the future.

P1 of Sweet Potato Feathery Mottle Virus Shows Strong Adaptation Capacity, Replacing P1-HCPro in a Chimeric Plum Pox Virus.

Potyviridae is the largest family of plant RNA viruses. Their genomes are expressed through long polyproteins that are usually headed by the leader endopeptidase P1. This protein can be classified as type A or type B based on host proteolytic requirements and RNA silencing suppression (RSS) capacity. The main Potyviridae genus is Potyvirus, and a group of potyviruses infecting sweet potato presents an enlarged P1 protein with a polymerase slippage motif that produces an extra product termed P1N-PISPO. These two proteins display some RSS activity and are expressed followed by HCPro, which appears to be the main RNA silencing suppressor in these viruses. Here, we studied the behavior of the P1 protein of Sweet potato feathery mottle virus (SPFMV) using a viral system based on a canonical potyvirus, Plum pox virus (PPV), and discovered that this protein is able to replace both PPV P1 and HCPro. We also found that P1N-PISPO, produced after polymerase slippage, provides extra RNA silencing suppression capacity to SPFMV P1 in this viral context. In addition, the results showed that presence of two type A P1 proteins was detrimental for viral viability. The ample recombination spectrum that we found in the recovered viruses supports the strong adaptation capacity of P1 proteins and signals the N-terminal part of SPFMV P1 as essential for RSS activity. Further analyses provided data to add extra layers to the evolutionary history of sweet potato-infecting potyvirids. IMPORTANCE Plant viruses represent a major challenge for agriculture worldwide and Potyviridae, being the largest family of plant RNA viruses, is one of the primary players. P1, the leader endopeptidase, is a multifunctional protein that contributes to the successful spread of these viruses over a wide host range. Understanding how P1 proteins work, their dynamic interplay during viral infection, and their evolutionary path is critical for the development of strategic tools to fight the multiple diseases these viruses cause. We focused our efforts on the P1 protein of Sweet potato feathery mottle virus, which is coresponsible for the most devastating disease in sweet potato. The significance of our research is in understanding the capacity of this protein to perform several independent functions, using this knowledge to learn more about P1 proteins in general and the potyvirids infecting this host.

The UFSP2/UFMylation Pathway Is Involved in Silica-Induced Pulmonary Injury.

Silicosis is an irreversible occupational pulmonary disease that is characterized as progressed pulmonary fibrosis. In this study, we investigated the changes of UFSP2 and the related UFMylation in silica-induced pulmonary injury mice models. The experimental silicosis models were prepared by intratracheal injection of silica particles, and the lung samples were harvested at the first or the seventh day after treatment. We found that the UFSP2 expression in the 1-day models was comparable, whereas it was upregulated in the 7-day models. Consistently, the UFMylation in the lung tissues of the 7-day models was activated. In addition, we observed the CADM2, an adhesion molecule, was reported to associate with epithelial-mesenchymal transition, was upregulated in the lungs of 7-day models. In contrast, it remained comparable in the 1-day models. Our data indicated that the UFSP2/UFMylation pathway and the CADM2 might be involved in the silica-induced pulmonary injury.

SENP1 is required for the growth, migration, and survival of human adipose-derived stem cells.

Human adipose-derived stem cells (hADSCs) are adult mesenchymal cells that have attracted the interest of clinical scientists and surgeons due to their large number of advantages including ease of access and expansion, abundance in cell culture, high proliferative rates, and lower senescence. SUMO/sentrin specific protease 1 (SENP1) is a critical protease that is required during the process of SUMOylation and deSUMOylation, which are dynamic mechanisms that influence cell cycle progression, cell proliferation, and apoptotic status. However, the contribution of SENP1 to these important cellular processes in hADSCs is largely uncharacterized and further studies in this area are required. Here, we show for the first time that after knock out SENP1 in hADSCs, their capacity to migrate and proliferate were inhibited, while apoptosis was enhanced. However, SENP1 did not significantly influence the morphology and MSC-related phenotypes of the hADSCs. These results highlight a role for SENP1 during hADSC growth, and its potential as a therapeutic target to improve the efficacy and safety of hADSCs in the clinic.

Evaluation of NDEL1 oligopeptidase activity in blood and brain in an animal model of schizophrenia: effects of psychostimulants and antipsychotics.

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.

HMGB1 mediates homocysteine-induced endothelial cells pyroptosis via cathepsin V-dependent pathway.

Endothelial cells injury and pro-inflammation cytokines release are the initial steps of hyperhomocysteinemia (HHcy)-associated vascular inflammation. Pyroptosis is a newly identified pro-inflammation form of programmed cell death, causing cell lysis and IL-1ß release, and characterized by the caspases-induced cleavage of its effector molecule gasdermins (GSDMs). However, the effect of homocysteine (Hcy) on endothelial cells pyroptosis and the underlying mechanisms have not been fully defined. We have previously reported that Hcy induces vascular endothelial inflammation accompanied by the increase of high mobility group box-1 protein (HMGB1) and lysosomal cysteine protease cathepsin V in endothelial cells, and other studies have shown that HMGB1 or cathepsins are involved in activation of NLRP3 inflammasome and caspase-1. Here, we investigated the role of HMGB1 and cathepsin V in the process of Hcy-induced pyroptosis. We observed an increase in plasma IL-1ß levels in HHcy patients and mice models, cathepsin V inhibitor reduced the plasma IL-1ß levels and cleavage of GSDMD full-length into GSDMD N-terminal in the thoracic aorta of hyperhomocysteinemia mice. Using cultured HUVECs, we observed that Hcy promoted GSDMD N-terminal expression, silencing GSDMD or HMGB1 rescued Hcy-induced pyroptosis. HMGB1 also increased GSDMD N-terminal expression, and silencing cathepsin V reversed HMGB1-induced pyroptosis. HMGB1 could increase lysosome permeability, and silencing cathepsin V attenuated HMGB1-induced activation of caspase-1. In conclusion, this study has delineated a novel mechanism that HMGB1 mediated Hcy-induced endothelial cells pyroptosis partly via cathepsin V-dependent pathway.

The role of radiation induced oxidative stress as a regulator of radio-adaptive responses.

Purpose: Various sources of radiation including radiofrequency, electromagnetic radiation (EMR), low- dose X-radiation, low-level microwave radiation and ionizing radiation (IR) are indispensable parts of modern life. In the current review, we discussed the adaptive responses of biological systems to radiation with a focus on the impacts of radiation-induced oxidative stress (RIOS) and its molecular downstream signaling pathways.Materials and methods: A comprehensive search was conducted in Web of Sciences, PubMed, Scopus, Google Scholar, Embase, and Cochrane Library. Keywords included Mesh terms of "radiation," "electromagnetic radiation," "adaptive immunity," "oxidative stress," and "immune checkpoints." Manuscripts published up until December 2019 were included.Results: RIOS induces various molecular adaptors connected with adaptive responses in radiation exposed cells. One of these adaptors includes p53 which promotes various cellular signaling pathways. RIOS also activates the intrinsic apoptotic pathway by depolarization of the mitochondrial membrane potential and activating the caspase apoptotic cascade. RIOS is also involved in radiation-induced proliferative responses through interaction with mitogen-activated protein kinases (MAPks) including p38 MAPK, ERK, and c-Jun N-terminal kinase (JNK). Protein kinase B (Akt)/phosphoinositide 3-kinase (PI3K) signaling pathway has also been reported to be involved in RIOS-induced proliferative responses. Furthermore, RIOS promotes genetic instability by introducing DNA structural and epigenetic alterations, as well as attenuating DNA repair mechanisms. Inflammatory transcription factors including macrophage migration inhibitory factor (MIF), nuclear factor κB (NF-κB), and signal transducer and activator of transcription-3 (STAT-3) paly major role in RIOS-induced inflammation.Conclusion: In conclusion, RIOS considerably contributes to radiation induced adaptive responses. Other possible molecular adaptors modulating RIOS-induced responses are yet to be divulged in future studies.

On ATG4B as Drug Target for Treatment of Solid Tumours-The Knowns and the Unknowns.

Autophagy is an evolutionary conserved stress survival pathway that has been shown to play an important role in the initiation, progression, and metastasis of multiple cancers; however, little progress has been made to date in translation of basic research to clinical application. This is partially due to an incomplete understanding of the role of autophagy in the different stages of cancer, and also to an incomplete assessment of potential drug targets in the autophagy pathway. While drug discovery efforts are on-going to target enzymes involved in the initiation phase of the autophagosome, e.g., unc51-like autophagy activating kinase (ULK)1/2, vacuolar protein sorting 34 (Vps34), and autophagy-related (ATG)7, we propose that the cysteine protease ATG4B is a bona fide drug target for the development of anti-cancer treatments. In this review, we highlight some of the recent advances in our understanding of the role of ATG4B in autophagy and its relevance to cancer, and perform a critical evaluation of ATG4B as a druggable cancer target.

Evaluation of clan CD C11 peptidase PNT1 and other Leishmania mexicana cysteine peptidases as potential drug targets.

Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is an urgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role in pathogenicity and virulence in Leishmania spp. and are promising targets for developing new anti-leishmanial drugs. Genetic drug target validation has been performed on a number of cysteine peptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidases for gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertain their cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1, LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. The other peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51, C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant (Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant of PNT1 (Δpnt1: PNT1flox/Δ pnt1:HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellum and on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits on the cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutant PNT1C134A, was lethal, suggesting that active PNT1 peptidase is required for parasite survival. Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidases that are potential drug targets in Leishmania.

Alcohol sedation in adult Drosophila is regulated by Cysteine proteinase-1 in cortex glia.

Although numerous studies have demonstrated that neuronal mechanisms regulate alcohol-related behaviors, very few have investigated the direct role of glia in behavioral responses to alcohol. The results described here begin to fill this gap in the alcohol behavior and gliobiology fields. Since Drosophila exhibit conserved behavioral responses to alcohol and their CNS glia are similar to mammalian CNS glia, we used Drosophila to begin exploring the role of glia in alcohol behavior. We found that knockdown of Cysteine proteinase-1 (Cp1) in glia increased Drosophila alcohol sedation and that this effect was specific to cortex glia and adulthood. These data implicate Cp1 and cortex glia in alcohol-related behaviors. Cortex glia are functionally homologous to mammalian astrocytes and Cp1 is orthologous to mammalian Cathepsin L. Our studies raise the possibility that cathepsins may influence behavioral responses to alcohol in mammals via roles in astrocytes.

Sortase A-mediated modification of the Streptococcus mutans transcriptome and virulence traits.

Sortase A contributes to adhesion and biofilm formation of Streptococcus mutans by anchoring surface proteins like P1 onto the cell wall, and few other functional characterization has been annotated to this protein and its coding gene srtA. In this study we investigated that whether srtA deletion would affect S. mutans virulence determinants in addition to adhesion and further explored whether these effects were caused due to changes in S. mutans genomic transcription. We used acid-killing assays, glycolytic rate assessments, and exopolysaccharide (EPS) formation tests to detect whether srtA deletion influenced S. mutans acid tolerance/production and glucan formation. Comparisons between RNA-sequencing data from both the exponential and stationary phases of UA159 and the srtA-deleted strain were made to determine the impact of srtA knockout on S. mutans genomic transcription. Results of our assays indicated that S. mutans aciduricity was enhanced in the srtA deleted strain when bacterial cells were directly subjected to pH 2.8, but the enhancement was repressed when the acid tolerance response was induced in advance. The srtA mutation strain exhibited reduced EPS formation in mature biofilms. SrtA deletion led to pleiotropic changes in the S. mutans transcriptome with a growth phase-dependent pattern. The affected genes mainly included those involved in aciduricity, carbohydrate transport, and EPS formation. It was concluded that S. mutans srtA exhibited multiple effects on the virulence traits of this pathogen, including acid tolerance and glucan formation, and that these alterations could be partially due to transcriptional changes upon loss of srtA.

The SUMO-Specific Protease Senp2 Regulates SUMOylation, Expression and Function of Human Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3) plays a vital role in removing a broad array of anionic drugs from kidney, thereby avoiding their possibly toxic side effects in the body. We earlier demonstrated that OAT3 is subjected to a specific type of post-translational modification called SUMOylation. SUMOylation is a dynamic event, where de-SUMOylation is catalyzed by a class of SUMO-specific proteases. In the present investigation, we assessed the role of SUMO-specific protease Senp2 in OAT3 SUMOylation, expression and function. We report here that overexpression of Senp2 in COS-7 cells led to a reduced OAT3 SUMOylation, which correlated well with a decreased OAT3 expression and transport activity. Such phenomenon was not observed in cells overexpressing an inactive mutant of Senp2. Furthermore, transfection of cells with Senp2-specific siRNA to knockdown the endogenous Senp2 resulted in an increased OAT3 SUMOylation, which correlated well with an enhanced OAT3 expression and transport activity. Coimmunoprecipitation experiments showed that Senp2 directly interacted with OAT3 in the kidneys of rats. Together these results provided first demonstration that Senp2 is a significant regulator for OAT3-mediated organic anion/drug transport.

Maturation of coagulation factor IX during Xase formation as deduced using factor VIII-derived peptides.

Blood coagulation involves extrinsic and intrinsic pathways, which merge at the activation step of blood coagulation factor X to factor Xa. This step is catalysed by the extrinsic or intrinsic Xase, which consists of a complex of factor VIIa and its cofactor tissue factor or factor IXa (FIXa) and its cofactor coagulation factor VIIIa (FVIIIa). Upon complex formation with FVIIIa, FIXa is conformationally activated to the Xase complex. However, the mechanistic understanding of this molecular recognition is limited. Here, we examined FVIIIa-FIXa binding in the context of FIXa's activation status. Given the complexity and the labile nature of FVIIIa, we decided to employ two FVIII-derived peptides (558-loop, a2 peptide) to model the cofactor binding of FIX(a) using biosensor chip technology. These two FVIII peptides are known to mediate the key interactions between FVIIIa and FIXa. We found both of these cofactor mimetics as well as full-length FVIIIa bind more tightly to zymogenic FIX than to proteolytically activated FIXa. Consequently and surprisingly, we observed that the catalytically inactive FIX zymogen can outcompete the activated FIXa from the complex with FVIIIa, resulting in an inactive, zymogenic Xase complex. By contrast, the thrombophilic Padua mutant FIXa-R170 in complex with the protein-substrate analogue BPTI bound tighter to FVIIIa than to the zymogen form FIX-R170L, suggesting that the active Xase complex preferentially forms in the Padua variant. Together, these results provide a mechanistic basis for the thrombophilic nature of the FIX-R170L mutant and suggest the existence of a newly discovered safety measure within the coagulation cascade.

OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes.

Astrocytes are critical regulators of neuroinflammation in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Growing evidence indicates that ubiquitination of signaling molecules is an important cell-intrinsic mechanism governing astrocyte function during MS and EAE Here, we identified an upregulation of the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) in astrocytes during MS and EAE Mice with astrocyte-specific OTUB1 ablation developed more severe EAE due to increased leukocyte accumulation, proinflammatory gene transcription, and demyelination in the spinal cord as compared to control mice. OTUB1-deficient astrocytes were hyperactivated in response to IFN-γ, a fingerprint cytokine of encephalitogenic T cells, and produced more proinflammatory cytokines and chemokines than control astrocytes. Mechanistically, OTUB1 inhibited IFN-γ-induced Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling by K48 deubiquitination and stabilization of the JAK2 inhibitor suppressor of cytokine signaling 1 (SOCS1). Thus, astrocyte-specific OTUB1 is a critical inhibitor of neuroinflammation in CNS autoimmunity.

A comprehensive in silico analysis of sortase superfamily.

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.