Lipopolysaccharide Primes Human Macrophages for Noncanonical Inflammasome-Induced Extracellular Vesicle Secretion.

Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs.

A homozygous POLR1A variant causes leukodystrophy and affects protein homeostasis.

RNA polymerase I transcribes ribosomal DNA to produce precursor 47S rRNA. Post-transcriptional processing of this rRNA generates mature 28S, 18S and 5.8S rRNAs, which form the ribosomes, together with 5S rRNA, assembly factors and ribosomal proteins. We previously reported a homozygous variant in the catalytic subunit of RNA polymerase I, POLR1A, in two brothers with leukodystrophy and progressive course. However, the disease mechanism remained unknown. In this report, we describe another missense variant POLR1A NM_015425.3:c.1925C>A; p.(Thr642Asn) in homozygosity in two unrelated patients. Patient 1 was a 16-year-old male and Patient 2 was a 2-year-old female. Both patients manifested neurological deficits, with brain MRIs showing hypomyelinating leukodystrophy and cerebellar atrophy; and in Patient 1 additionally with hypointensity of globi pallidi and small volume of the basal ganglia. Patient 1 had progressive disease course, leading to death at the age of 16.5 years. Extensive in vitro experiments in fibroblasts from Patient 1 documented that the mutated POLR1A led to aberrant rRNA processing and degradation, and abnormal nucleolar homeostasis. Proteomics data analyses and further in vitro experiments documented abnormal protein homeostasis, and endoplasmic reticulum stress responses. We confirm that POLR1A biallelic variants cause neurodegenerative disease, expand the knowledge of the clinical phenotype of the disorder, and provide evidence for possible pathological mechanisms leading to POLR1A-related leukodystrophy.

Tyr192 Regulates Lymphocyte-Specific Tyrosine Kinase Activity in T Cells.

TCR signaling critically depends on the tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase). Two phosphotyrosines, the activating pTyr394 and the inhibitory pTyr505, control Lck activity. Recently, pTyr192 in the Lck SH2 domain emerged as a third regulator. How pTyr192 may affect Lck function remains unclear. In this study, we explored the role of Lck Tyr192 using CRISPR/Cas9-targeted knock-in mutations in the human Jurkat T cell line. Our data reveal that both Lck pTyr394 and pTyr505 are controlled by Lck Tyr192 Lck with a nonphosphorylated SH2 domain (Lck Phe192) displayed hyperactivity, possibly by promoting Lck Tyr394 transphosphorylation. Lck Glu192 mimicking stable Lck pTyr192 was inhibited by Tyr505 hyperphosphorylation. To overcome this effect, we further mutated Tyr505 The resulting Lck Glu192/Phe505 displayed strongly increased amounts of pTyr394 both in resting and activated T cells. Our results suggest that a fundamental role of Lck pTyr192 may be to protect Lck pTyr394 and/or pTyr505 to maintain a pool of already active Lck in resting T cells. This provides an additional mechanism for fine-tuning of Lck as well as T cell activity.

Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer.

BACKGROUND: Targeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L. METHODS: We combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes. RESULTS: Dot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine-protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells. CONCLUSIONS: Identification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

Clinical characteristics and proteome modifications in two Charcot-Marie-Tooth families with the AARS1 Arg326Trp mutation.

BACKGROUND: Aminoacyl tRNA-synthetases are ubiquitously-expressed enzymes that attach amino acids to their cognate tRNA molecules. Mutations in several genes encoding aminoacyl tRNA-synthetases, have been associated with peripheral neuropathy, i.e. AARS1, GARS1, HARS1, YARS1 and WARS1. The pathogenic mechanism underlying AARS1-related neuropathy is not known. METHODS: From 2012 onward, all probands presenting at Telemark Hospital (Skien, Norway) with peripheral neuropathy were screened for variants in AARS1 using an "in-house" next-generation sequencing panel. DNA from patient's family members was examined by Sanger sequencing. Blood from affected family members and healthy controls were used for quantification of AARS1 mRNA and alanine. Proteomic analyses were conducted in peripheral blood mononuclear cells (PBMC) from four affected family members and five healthy controls. RESULTS: Seventeen individuals in two Norwegian families affected by Charcot-Marie-Tooth disease (CMT) were characterized in this study. The heterozygous NM_001605.2:c.976C > T p.(Arg326Trp) AARS1 mutation was identified in ten affected family members. All living carriers had a mild to severe length-dependent sensorimotor neuropathy. Three deceased obligate carriers aged 74-98 were reported to be unaffected, but were not examined in the clinic. Proteomic studies in PBMC from four affected individuals suggest an effect on the immune system mediated by components of a systemic response to chronic injury and inflammation. Furthermore, altered expression of proteins linked to mitochondrial function/dysfunction was observed. Proteomic data are available via ProteomeXchange using identifier PXD023842. CONCLUSION: This study describes clinical and neurophysiological features linked to the p.(Arg326Trp) variant of AARS1 in CMT-affected members of two Norwegian families. Proteomic analyses based on of PBMC from four CMT-affected individuals suggest that involvement of inflammation and mitochondrial dysfunction might contribute to AARS1 variant-associated peripheral neuropathy.

Function and Regulation of Noncanonical Caspase-4/5/11 Inflammasome.

Inflammasomes are multiprotein complexes of the innate immune system that orchestrate development of inflammation by activating the secretion of proinflammatory cytokines, IL-1ß and IL-18. The LPS of Gram-negative bacteria have been shown to activate a novel, noncanonical inflammasome by directly binding in the cytosol to human caspase-4 and mouse caspase-11. Activation of noncanonical inflammasome exerts two major effects: it activates the NLRP3-caspase-1-mediated processing and secretion of IL-1ß and IL-18 and induces the inflammatory cell death, pyroptosis, via gasdermin D. This previously unexpected cytosolic LPS sensing of the innate immune system provides critical hints for host response to Gram-negative bacterial infections and development of different inflammatory diseases. However, many of its molecular regulatory mechanisms are yet to be discovered. In this review, we provide comprehensive analysis of current understanding of intracellular LPS detection and pyroptosis via noncanonical inflammasome and discuss the recently proposed mechanisms of its function and regulation.

Interaction Proteomics Identifies ERbeta Association with Chromatin Repressive Complexes to Inhibit Cholesterol Biosynthesis and Exert An Oncosuppressive Role in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by poor response to therapy and low overall patient survival. Recently, Estrogen Receptor beta (ERß) has been found to be expressed in a fraction of TNBCs where, because of its oncosuppressive actions on the genome, it represents a potential therapeutic target, provided a better understanding of its actions in these tumors becomes available. To this end, the cell lines Hs 578T, MDA-MB-468 and HCC1806, representing the claudin-low, basal-like 1 and 2 TNBC molecular subtypes respectively, were engineered to express ERß under the control of a Tetracycline-inducible promoter and used to investigate the effects of this transcription factor on gene activity. The antiproliferative effects of ERß in these cells were confirmed by multiple functional approaches, including transcriptome profiling and global mapping of receptor binding sites in the genome, that revealed direct negative regulation by ERß of genes, encoding for key components of cellular pathways associated to TNBC aggressiveness representing novel therapeutic targets such as angiogenesis, invasion, metastasis and cholesterol biosynthesis. Supporting these results, interaction proteomics by immunoprecipitation coupled to nano LC-MS/MS mass spectrometry revealed ERß association with several potential nuclear protein partners, including key components of regulatory complexes known to control chromatin remodeling, transcriptional and post-transcriptional gene regulation and RNA splicing. Among these, ERß association with the Polycomb Repressor Complexes 1 and 2 (PRC1/2), known for their central role in gene regulation in cancer cells, was confirmed in all three TNBC subtypes investigated, suggesting its occurrence independently from the cellular context. These results demonstrate a significant impact of ERß in TNBC genome activity mediated by its cooperation with regulatory multiprotein chromatin remodeling complexes, providing novel ground to devise new strategies for the treatment of these diseases based on ligands affecting the activity of this nuclear receptor or some of its protein partners.

Immune complexes, innate immunity, and NETosis in ChAdOx1 vaccine-induced thrombocytopenia.

AIMS: We recently reported five cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) 7-10 days after receiving the first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine against corona virus disease 2019 (COVID-19). We aimed to investigate the pathogenic immunological responses operating in these patients. METHODS AND RESULTS: We assessed circulating inflammatory markers by immune assays and immune cell phenotyping by flow cytometry analyses and performed immunoprecipitation with anti-platelet factor (PF)4 antibody in plasma samples followed by mass spectrometry from all five patients. A thrombus was retrieved from the sinus sagittal superior of one patient and analysed by immunohistochemistry and flow cytometry. Precipitated immune complexes revealed multiple innate immune pathway triggers for platelet and leucocyte activation. Plasma contained increased levels of innate immune response cytokines and markers of systemic inflammation, extensive degranulation of neutrophils, and tissue and endothelial damage. Blood analyses showed activation of neutrophils and increased levels of circulating H3Cit, dsDNA, and myeloperoxidase-DNA complex. The thrombus had extensive infiltration of neutrophils, formation of neutrophil extracellular traps (NETs), and IgG deposits. CONCLUSIONS: The results show that anti-PF4/polyanion IgG-mediated thrombus formation in VITT patients is accompanied by a massive innate immune activation and particularly the fulminant activation of neutrophils including NETosis. These results provide novel data on the immune response in this rare adenoviral vector-induced VITT.

A high-throughput pipeline for validation of antibodies.

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

Identification of Antiestrogen-Bound Estrogen Receptor α Interactomes in Hormone-Responsive Human Breast Cancer Cell Nuclei.

Estrogen receptor alpha (ERα) is a ligand-inducible transcription factor which mediates estrogen actions in hormone-responsive tumors and is targeted by effective anticancer therapies based on the ERα antagonist ligands, selective estrogen receptor modulators (such as Tamoxifen/TAM) or disruptors (such as Fulvestrant/ICI). Despite its importance for cancer therapy, including acquired resistance to endocrine therapy, the molecular basis of ERα response to different ligands is not fully known to date. Interaction proteomics shows great potential to identify and characterize molecular mechanisms of disease based on physical and functional protein-protein interaction networks. Tandem affinity purification coupled to mass spectrometry is applied here for mapping in hormone-responsive breast cancer cells nuclei, the ERα interactomes, induced by each of the two classes of antiestrogens. The results provide new insights on the molecular bases for antiestrogen-mediated control of ERα function and reveal new potential ways to overcome endocrine therapy resistance in cancer.

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia.

Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.

The cardiac syndecan-4 interactome reveals a role for syndecan-4 in nuclear translocation of muscle LIM protein (MLP).

Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart.

HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Pancreatic cancer cells show lower oleic acid oxidation and their conditioned medium inhibits oleic acid oxidation in human myotubes.

BACKGROUND: /Objectives: We aimed to metabolically compare healthy primary human pancreatic epithelial cells (hPEC) to a pancreatic cancer cell line (PANC-1) and explore the effect on energy metabolism of exposing primary human myotubes to conditioned medium from hPEC and PANC-1 cells. METHODS: Differences in metabolism were examined with radiolabeled glucose, oleic acid and lactic acid, and by qPCR. Mass spectrometry-based proteomics was used to study global protein secretion from the two cell types. Pathway analyses were performed. RESULTS: PANC-1 cells tended to have higher glucose uptake, production of lactic acid, and glucose oxidation compared to hPEC cells. PANC-1 cells had higher uptake but lower oxidation of oleic acid, and mitochondrial reserve capacity from oleic acid was lower in PANC-1 cells. These differences in energy metabolism were reflected by differences in gene expressions and pathway analyses of the secretome. Conditioned medium from PANC-1 cells attenuated oleic acid oxidation in primary human myotubes. CONCLUSIONS: Metabolic characterization of the PANC-1 cells revealed a glycolytic phenotype since they had an active glucose oxidation. Furthermore, PANC-1 cells showed a lower oleic acid oxidation and secreted a high amount of proteins into conditioned medium that also induced a reduced oleic acid oxidation in myotubes.

Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation.

Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, adisintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.

Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

Invading pathogens provoke robust innate immune responses in Dipteran insects, such as Drosophila melanogaster In a systemic bacterial infection, a humoral response is induced in the fat body. Gram-positive bacteria trigger the Toll signaling pathway, whereas gram-negative bacterial infections are signaled via the immune deficiency (IMD) pathway. We show here that the RNA interference-mediated silencing of Furin1-a member of the proprotein convertase enzyme family-specifically in the fat body, results in a reduction in the expression of antimicrobial peptides. This, in turn, compromises the survival of adult fruit flies in systemic infections that are caused by both gram-positive and -negative bacteria. Furin1 plays a nonredundant role in the regulation of immune responses, as silencing of Furin2, the other member of the enzyme family, had no effect on survival or the expression of antimicrobial peptides upon a systemic infection. Furin1 does not directly affect the Toll or IMD signaling pathways, but the reduced expression of Furin1 up-regulates stress response factors in the fat body. We also demonstrate that Furin1 is a negative regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway, which is implicated in stress responses in the fly. In summary, our data identify Furin1 as a novel regulator of humoral immunity and cellular stress responses in Drosophila-Aittomäki, S., Valanne, S., Lehtinen, T., Matikainen, S., Nyman, T. A., Rämet, M., Pesu, M. Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.-Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.

Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages.

Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1ß. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1ß and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.

Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion.

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane.

Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.