Ubiquilin 1 Promotes IFN-?-Induced Xenophagy of Mycobacterium tuberculosis.
ABSTRACT: The success of Mycobacterium tuberculosis (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-?) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-?-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-? activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-?. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.
Project description:Although the aetiology of amyotrophic lateral sclerosis (ALS) remains poorly understood, impaired proteostasis is a common feature of different forms of ALS. Mutations in genes encoding ubiquilins, UBQLN2 and UBQLN4, cause familial ALS. The role of ubiquilins in proteasomal degradation is well established, but their role in autophagy-lysosomal clearance is poorly defined. Here, we describe a crosstalk between endoplasmic reticulum stress, mTOR signalling and autophagic flux in Drosophila and mammalian cells lacking ubiquilins. We found that loss of ubiquilins leads to endoplasmic reticulum stress, impairs mTORC1 activity, promotes autophagy and causes the demise of neurons. We show that ubiquilin mutants display defective autophagic flux due to reduced lysosome acidification. Ubiquilins are required to maintain proper levels of the V0a/V100 subunit of the vacuolar H+-ATPase and lysosomal pH. Feeding flies acidic nanoparticles alleviates defective autophagic flux in ubiquilin mutants. Hence, our studies reveal a conserved role for ubiquilins as regulators of autophagy by controlling vacuolar H+-ATPase activity and mTOR signalling.
Project description:The acclimatization of brown adipose tissue (BAT) to sustained cold exposure requires an adaptive increase in proteasomal protein quality control. Ubiquilins represent a recently identified family of shuttle proteins with versatile functions in protein degradation, such as facilitating substrate targeting and proteasomal degradation. However, whether ubiquilins participate in brown adipocyte function has not been investigated so far. Here, we determine the role of ubiquilins for proteostasis and non-shivering thermogenesis in brown adipocytes. We found that <i>Ubqln1, 2</i> and <i>4</i> are highly expressed in BAT and their expression was induced by cold and proteasomal inhibition. Surprisingly, silencing of ubiquilin gene expression (one or multiple in combinations) did not lead to aggravated ER stress or inflammation. Moreover, ubiquitin level and proteasomal activity under basal conditions were not impacted by loss of ubiquilins. Also, non-shivering thermogenesis measured by norepinephrine-induced respiration remained intact after loss of ubiquilins. In conclusion, ubiquilin proteins are highly abundant in BAT and regulated by cold, but they are dispensable for brown adipocyte proteostasis and thermogenesis.
Project description:Ubiquilins (Ubqlns)-a family of ubiquitin-binding proteins-are involved in several protein degradation pathways and have been implicated in various neurodegenerative diseases. Ubqln1 regulates autophagosome maturation during autophagy-mediated degradation. We now show that Ubqln4 mediates the interaction between Ubqln1 and the autophagy machinery by recruiting Ubqln1 to LC3. This targeting of Ubqln1 to autophagosomes requires the Ubqln4 UBL domain and the Ubqln1 UBA domain. This study identifies a new role for Ubqln4, expanding the role for Ubqlns in protein degradation.
Project description:BACKGROUND: Ubiquilins are proteins that function as ubiquitin receptors in eukaryotes. Mutations in two ubiquilin-encoding genes have been linked to the genesis of neurodegenerative diseases. However, ubiquilin functions are still poorly understood. RESULTS: In this study, evolutionary and functional data are combined to determine the origin and diversification of the ubiquilin gene family and to characterize novel potential roles of ubiquilins in mammalian species, including humans. The analysis of more than six hundred sequences allowed characterizing ubiquilin diversity in all the main eukaryotic groups. Many organisms (e. g. fungi, many animals) have single ubiquilin genes, but duplications in animal, plant, alveolate and excavate species are described. Seven different ubiquilins have been detected in vertebrates. Two of them, here called UBQLN5 and UBQLN6, had not been hitherto described. Significantly, marsupial and eutherian mammals have the most complex ubiquilin gene families, composed of up to 6 genes. This exceptional mammalian-specific expansion is the result of the recent emergence of four new genes, three of them (UBQLN3, UBQLN5 and UBQLNL) with precise testis-specific expression patterns that indicate roles in the postmeiotic stages of spermatogenesis. A gene with related features has independently arisen in species of the Drosophila genus. Positive selection acting on some mammalian ubiquilins has been detected. CONCLUSIONS: The ubiquilin gene family is highly conserved in eukaryotes. The infrequent lineage-specific amplifications observed may be linked to the emergence of novel functions in particular tissues.
Project description:Comparative phosphoproteomics of <i>Mycobacterium tuberculosis</i> (Mtb)- and <i>Mycobacterium bovis</i> BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylation status, herein, we compared the phosphorylation profile of macrophages upon exposure to Mtb and BCG. We observed a significant dephosphorylation of proteins following Mtb infection relative to those with uninfected or BCG-infected cells. A comprehensive tandem mass tag mass spectrometry (MS) approach detected ∼10% phosphosites on a variety of host proteins that are modulated in response to infection. Interestingly, the innate immune-enhancing interferon (IFN)-stimulated genes were identified as a class of proteins differentially phosphorylated during infection, including the cytosolic RNA sensor RIG-I, which has been implicated in the immune response to bacterial infection. We show that Mtb infection results in the activation of RIG-I in primary human macrophages. Studies using RIG-I knockout macrophages reveal that the Mtb-mediated activation of RIG-I promotes IFN-β, IL-1α, and IL-1β levels, dampens autophagy, and facilitates intracellular Mtb survival. To our knowledge, this is the first study providing exhaustive information on relative and quantitative changes in the global phosphoproteome profile of host macrophages that can be further explored in designing novel anti-TB drug targets. The peptide identification and MS/MS spectra have been deposited to the ProteomeXchange Consortium <i>via</i> the PRIDE partner repository with the dataset identifier PXD013171.
Project description:As a very successful pathogen with outstanding adaptive properties, <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) has developed a plethora of sophisticated mechanisms to subvert host defenses and effectively enter and replicate in the harmful environment inside professional phagocytes, namely, macrophages. Here, we demonstrated the binding interaction of <i>Mtb</i> with a major human acute phase protein, namely, serum amyloid A (SAA1), and identified AtpA (Rv1308), ABC (Rv2477c), EspB (Rv3881c), TB 18.6 (Rv2140c), and ThiC (Rv0423c) membrane proteins as mycobacterial effectors responsible for the pathogen-host protein interplay. SAA1-opsonization of <i>Mtb</i> prior to the infection of human macrophages favored bacterial entry into target phagocytes accompanied by a substantial increase in the load of intracellularly multiplying and surviving bacteria. Furthermore, binding of human SAA1 by <i>Mtb</i> resulted in the up- or downregulation of the transcriptional response of tubercle bacilli. The most substantial changes were related to the increased expression level of the genes of two operons encoding mycobacterial transporter systems, namely, <i>mmpL5/mmpS5</i> (<i>rv0676c</i>), and <i>rv1217c</i>, <i>rv1218c</i>. Therefore, we postulate that during infection, <i>Mtb</i>-SAA1 binding promotes the infection of host macrophages by tubercle bacilli and modulates the functional response of the pathogen.
Project description:Ubiquilin‑1 (UBQLN1) is an essential factor for the maintenance of proteostasis in cells. It is important for the regulation of different protein degradation mechanisms, including the ubiquitin‑proteasome system, autophagy and endoplasmic reticulum‑associated protein degradation pathways. However, the role of UBQLN1 in cancer progression remains largely unknown. In the present study, the expression, functions and molecular mechanisms of UBQLN1 in breast cancer tissue samples and cell lines were explored. Immunohistochemical and bioinformatics analyses revealed that UBQLN1 expression was significantly upregulated in breast cancer tissues and cell lines. UBQLN1 expression in breast cancer was significantly associated with lymph node metastasis and TNM stage. Moreover, a high UBQLN1 expression was a predictor of an unfavorable survival in patients with breast cancer. <i>In vitro</i>, UBQLN1 silencing markedly inhibited cell migration and invasion, epithelial‑to‑mesenchymal transition (EMT) and MMP expression. UBQLN1 silencing attenuated the stem cell‑like properties of breast cancer cells, including their mammosphere‑forming abilities. UBQLN1 knockdown also enhanced breast cancer cell chemosensitivity to paclitaxel. The expression levels of the stem cell markers. Aldehyde dehydrogenase 1 (ALDH1), Oct‑4 and Sox2 were significantly decreased in the cells in which UBQLN1 was silenced, whereas breast cancer stem cells exhibited an increased expression of UBQLN1. Mechanistically, UBQLN1 knockdown inhibited the activation of AKT signaling, as revealed by the increased PTEN expression and the decreased expression of phosphorylated AKT in cells in which UBQLN1 was silenced. On the whole, the present study demonstrates that UBQLN1 is aberrantly upregulated in breast cancer and predicts a poor prognosis. The silencing of UBQLN1 inhibited the invasion, EMT and stemness of breast cancer cells, possibly via AKT signaling.
Project description:We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions.
Project description:Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1-/- mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1-/- B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.
Project description:Iron is a crucial micronutrient for both mammals and their associated pathogens, and extensive literature has shown that Mycobacterium tuberculosis (Mtb) bacilli inhibited from acquiring iron from the host are severely attenuated. In contrast, increased dietary iron concentrations or patients with hemochromatosis have long been associated with a more severe tuberculosis (TB) disease outcome. We have observed that upon macrophage infection, Mtb bacilli strongly promote intracellular iron sequestration, both through increased expression of hepcidin, a key mammalian iron regulatory protein, and downregulation of the iron exporter protein, ferroportin. Heparin is a highly sulfated glycosaminoglycan released by mast cells and basophils at sites of tissue injury. During Mtb infection, heparin alters intracellular trafficking in alveolar epithelial cells and decreases extrapulmonary dissemination but recently, heparin also has been reported to inhibit hepcidin expression in hepatocytes, decreasing intracellular iron availability. In this report, we demonstrate that heparin significantly reduces hepcidin expression in macrophages infected with Mtb bacilli. Heparin-treated macrophages have higher ferroportin expression compared to untreated macrophages, promoting iron export and decreasing iron availability to intracellular bacilli. Thus, here we describe a novel immunomodulatory effect and potential therapeutic role for heparin against mycobacterial infection in human macrophages.