Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target.

Immunotherapy of PD-L1/PD-1 blockage elicited impressive clinical benefits for cancer treatment. However, the relative low response and therapy resistance highlight the need to better understand the molecular regulation of PD-L1 in tumors. Here, we report that PD-L1 is a target of UFMylation. UFMylation of PD-L1 destabilizes PD-L1 by synergizing its ubiquitination. Inhibition of PD-L1 UFMylation via silencing of UFL1 or Ubiquitin-fold modifier 1 (UFM1), or the defective UFMylation of PD-L1, stabilizes the PD-L1 in multiple human and murine cancer cells, and undermines antitumor immunity in vitro and mice, respectively. Clinically, UFL1 expression was decreased in multiple cancers and lower expression of UFL1 negatively correlated with the response of anti-PD1 therapy in melanoma patients. Moreover, we identified a covalent inhibitor of UFSP2 that promoted the UFMylation activity and contributed to the combination therapy with PD-1 blockade. Our findings identified a previously unrecognized regulator of PD-L1 and highlighted UFMylation as a potential therapeutic target.

UFMylation of HRD1 regulates endoplasmic reticulum homeostasis.

Ubiquitin fold modifier 1 is a small ubiquitin-like protein modifier that is essential for embryonic development of metazoans. Although UFMylation has been connected to endoplasmic reticulum homeostasis, the underlying mechanisms and the relevant cellular targets are largely unknown. Here, we show that HRD1, a ubiquitin ligase of ER-associated protein degradation (ERAD), is a novel substrate of UFM1 conjugation. HRD1 interacts with UFMylation components UFL1 and DDRGK1 and is UFMylated at Lys610 residue. In UFL1-depleted cells, the stability of HRD1 is increased and its ubiquitination modification is reduced. In the event of ER stress, the UFMylation and ubiquitination modification of HRD1 is gradually inhibited over time. Alteration of HRD1 Lys610 residue to arginine impairs its ability to degrade unfolded or misfolded proteins to disturb protein processing in ER. These results suggest that UFMylation of HRD1 facilitates ERAD function to maintain ER homeostasis.

TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment.

Telomerase plays a pivotal role in tumorigenesis by both telomere-dependent and telomere-independent activities, although the underlying mechanisms are not completely understood. Using single-sample gene set enrichment analysis (ssGSEA) across 9,264 tumour samples, we observe that expression of telomerase reverse transcriptase (TERT) is closely associated with immunosuppressive signatures. We demonstrate that TERT can activate a subclass of endogenous retroviruses (ERVs) independent of its telomerase activity to form double-stranded RNAs (dsRNAs), which are sensed by the RIG-1/MDA5-MAVS signalling pathway and trigger interferon signalling in cancer cells. Furthermore, we show that TERT-induced ERV/interferon signalling stimulates the expression of chemokines, including CXCL10, which induces the infiltration of suppressive T-cell populations with increased percentage of CD4+ and FOXP3+ cells. These data reveal an unanticipated role for telomerase as a transcriptional activator of ERVs and provide strong evidence that TERT-mediated ERV/interferon signalling contributes to immune suppression in tumours.

Human UFSP1 translated from an upstream near-cognate initiation codon functions as an active UFM1-specific protease.

Ubiquitin-fold modifier 1 (UFM1) is a recently identified ubiquitin-like posttranslational modification with important biological functions. However, the regulatory mechanisms governing UFM1 modification of target proteins (UFMylation) and the cellular processes controlled by UFMylation remain largely unknown. It has been previously shown that a UFM1-specific protease (UFSP2) mediates the maturation of the UFM1 precursor and drives the de-UFMylation reaction. Furthermore, it has long been thought that UFSP1, an ortholog of UFSP2, is inactive in many organisms, including human, because it lacks an apparent protease domain when translated from the canonical start codon (445AUG). Here, we demonstrate using the combination of site-directed mutagenesis, CRISPR/Cas9-mediated genome editing, and mass spectrometry approaches that translation of human UFSP1 initiates from an upstream near-cognate codon, 217CUG, via eukaryotic translation initiation factor eIF2A-mediated translational initiation rather than from the annotated 445AUG, revealing the presence of a catalytic protease domain containing a Cys active site. Moreover, we show that both UFSP1 and UFSP2 mediate maturation of UFM1 and de-UFMylation of target proteins. This study demonstrates that human UFSP1 functions as an active UFM1-specific protease, thus contributing to our understanding of the UFMylation/de-UFMylation process.

Human telomerase reverse transcriptase is a novel target of Hippo-YAP pathway.

Telomerase plays a pivotal role in tumorigenesis by maintaining telomere homeostasis, a hallmark of cancer. However, the mechanisms by which telomerase is reactivated or upregulated during tumorigenesis remain incompletely understood. Here, we report that the Hippo pathway effector Yes-associated protein (YAP) regulates the expression of human telomerase reverse transcriptase (hTERT). Ectopic expression or physiological activation of YAP increases hTERT expression, whereas knockdown of YAP decreases the expression of hTERT. YAP binds to the hTERT promoter through interaction with the TEA domain family transcription factors and activates hTERT transcription. Furthermore, sustained YAP hyperactivation promotes telomerase activity and extends telomere length, with increased hTERT expression. In addition, we show that hTERT expression is positively correlated with YAP activation in human liver cancer tissues. Together, our results demonstrate that YAP promotes hTERT expression, which could contribute to tumor progression.

MRE11 UFMylation promotes ATM activation.

A proper DNA damage response (DDR) is essential to maintain genome integrity and prevent tumorigenesis. DNA double-strand breaks (DSBs) are the most toxic DNA lesion and their repair is orchestrated by the ATM kinase. ATM is activated via the MRE11-RAD50-NBS1 (MRN) complex along with its autophosphorylation at S1981 and acetylation at K3106. Activated ATM rapidly phosphorylates a vast number of substrates in local chromatin, providing a scaffold for the assembly of higher-order complexes that can repair damaged DNA. While reversible ubiquitination has an important role in the DSB response, modification of the newly identified ubiquitin-like protein ubiquitin-fold modifier 1 and the function of UFMylation in the DDR is largely unknown. Here, we found that MRE11 is UFMylated on K282 and this UFMylation is required for the MRN complex formation under unperturbed conditions and DSB-induced optimal ATM activation, homologous recombination-mediated repair and genome integrity. A pathogenic mutation MRE11(G285C) identified in uterine endometrioid carcinoma exhibited a similar cellular phenotype as the UFMylation-defective mutant MRE11(K282R). Taken together, MRE11 UFMylation promotes ATM activation, DSB repair and genome stability, and potentially serves as a therapeutic target.

Role of telomerase in the tumour microenvironment.

Telomeres are specialized genomic structures that protect chromosomal ends to maintain genomic stability. Telomeric length is primarily regulated by the telomerase complex, essentially consisting of an RNA template (TERC), an enzymatic subunit (telomerase reverse transcriptase, TERT). In humans, telomerase activity is repressed during embryonic differentiation and is absent in most somatic cells. However, it is upregulated or reactivated in 80%-90% of the primary tumours in humans. The human TERT (hTERT) plays a pivotal role in cellular immortality and tumourigenesis. However, the molecular mechanisms of telomerase functioning in cancer have not been fully understood beyond the telomere maintenance. Several research groups, including ours, have demonstrated that hTERT possesses vital functions independent of its telomere maintenance, including angiogenesis, inflammation, cancer cell stemness, and epithelial-mesenchymal transformation (EMT). All these telomere-independent activities of hTERT may contribute to the regulation of the dynamics and homeostasis of the tumour microenvironment (TME), thereby promoting tumour growth and development. Cancer progression and metastasis largely depend upon the interactions between cancer cells and their microenvironment. In this review, the involvement of TERT in the tumour microenvironment and the underlying implications in cancer therapeutics have been summarized.

Ptrf transgenic mice exhibit obesity and fatty liver.

Polymerase I and transcript release factor (Ptrf, also known as Cavin1) is an essential component in the biogenesis and function of caveolae. Ptrf knockout mice or patients with PTRF mutations exhibit numerous pathologies including markedly aberrant fuel metabolism, lipodystrophy and muscular dystrophy. In this study, we generated Ptrf transgenic mice to explore its function in vivo. Compared with wild-type (WT) mice, we found that the Ptrf transgenic mice showed obesity with an increased level of ALT (alanine aminotransferase) and AST (aspartate transaminase). Ptrf transgenic mice exhibited severe fat degeneration and a higher degree of fat accumulation in the liver compared with WT mice. Consistently, we found that the expression of the fat synthesis gene, Fasn, was increased in the liver of Ptrf transgenic mice. Thus, Ptrf transgenic mice would be a good model for investigating the molecular mechanism and therapeutic targets of obesity and fatty liver associated diseases.

hTERT promotes tumor angiogenesis by activating VEGF via interactions with the Sp1 transcription factor.

Angiogenesis is recognized as an important hallmark of cancer. Although telomerase is thought to be involved in tumor angiogenesis, the evidence and underlying mechanism remain elusive. Here, we demonstrate that human telomerase reverse transcriptase (hTERT) activates vascular epithelial growth factor (VEGF) gene expression through interactions with the VEGF promoter and the transcription factor Sp1. hTERT binds to Sp1 in vitro and in vivo and stimulates angiogenesis in a manner dependent on Sp1. Deletion of the mTert gene in the first generation of Tert null mice compromised tumor growth, with reduced VEGF expression. In addition, we show that hTERT expression levels are positively correlated with those of VEGF in human gastric tumor samples. Together, our results demonstrate that hTERT facilitates tumor angiogenesis by up-regulating VEGF expression through direct interactions with the VEGF gene and the Sp1 transcription factor. These results provide novel insights into hTERT function in tumor progression in addition to its role in telomere maintenance.

Phosphatase Wip1 controls antigen-independent B-cell development in a p53-dependent manner.

Wild-type p53-induced phosphatase 1 (Wip1), a phosphatase previously considered as an oncogene, has been implicated in the regulation of thymus homeostasis and neutrophil maturation. However, the role of Wip1 in B-cell development is unknown. We show that Wip1-deficient mice exhibit a significant reduction of B-cell numbers in the bone marrow, peripheral blood, and spleen. A reciprocal transplantation approach revealed a cell-intrinsic defect in early B-cell precursors caused by Wip1 deficiency. Further experiments revealed that Wip1 deficiency led to a sustained activation of p53 in B cells, which led to increased level of apoptosis in the pre-B-cell compartment. Notably, the impairment of B-cell development in Wip1-deficient mice was completely rescued by genetic ablation of p53, but not p21. Therefore, loss of Wip1 phosphatase induces a p53-dependent, but p21-independent, mechanism that impairs B-cell development by enhancing apoptosis in early B-cell precursors. Moreover, Wip1 deficiency exacerbated a decline in B-cell development caused by aging as evidenced in mice with aging and mouse models with serial competitive bone marrow transplantation, respectively. Our present data indicate that Wip1 plays a critical role in maintaining antigen-independent B-cell development in the bone marrow and preventing an aging-related decline in B-cell development.

Human telomerase reverse transcriptase regulates MMP expression independently of telomerase activity via NF-κB-dependent transcription.

Telomerase plays a pivotal role in the pathology of aging and cancer by controlling telomere length and integrity. However, accumulating evidence indicates that telomerase reverse transcriptase may have fundamental biological functions independent of its enzymatic activity in telomere maintenance. In this study, the ectopic expression of human telomerase reverse transcriptase (hTERT) and its catalytic mutant hTERT K626A induced cancer cell invasion accompanied by the up-regulation of the metalloproteinases (MMPs) MMP1, -3, -9, and -10. Both hTERT and hTERT K626A induced MMP9 mRNA expression and promoter activity in an NF-κB-dependent manner. hTERT and hTERT K626A also regulated the expression of several NF-κB target genes in cancer cell lines. Furthermore, both hTERT and hTERT K626A interacted with NF-κB p65 and increased NF-κB p65 nuclear accumulation and DNA binding. A mammalian 1-hybrid assay showed a functional interplay between hTERT and NF-κB p65 that may mediate NF-κB-dependent transcription activation in cells. Together, these data reveal a telomere-independent role for telomerase as a transcriptional modulator of the NF-κB signaling pathway and a possible contributor to cancer development and progression.

Increased polymerase I and transcript release factor (Cavin-1) expression attenuates platelet-derived growth factor receptor signalling in senescent human fibroblasts.

1. Previously, we showed that the essential caveolar component polymerase I and transcript release factor (PTRF) was upregulated and promoted caveolae formation in senescent cells. In addition, we found that overexpression of PTRF increased the number of caveolae and induced cellular senescence. 2. Unresponsiveness to growth factor is one of the fundamental characteristics of senescent cells, although normal levels of receptors and downstream signalling molecules are present in senescent cells. 3. Herein, we investigated the role of PTRF in the regulation of platelet-derived growth factor (PDGF) signalling in young and senescent cells. 4. We first confirmed that PTRF was upregulated in senescent human fibroblasts and aged mouse tissues. We then examined the activation of extracellular signal-regulated kinases (ERK) in young and senescent cells after PDGF stimulation. 5. Our results show that expression of PDGF receptors (PDGFRs) was not altered during cellular senescence. Interestingly, phosphorylation of ERK1/2 was induced upon PDGF stimulation of young, replicating cells but not senescent cells. Induction of ERK1/2 phosphorylation was impaired in senescent cells and PTRF-overexpressing presenescent cells. Furthermore, our results show that PTRF interacts with PDGFRs and this interaction is increased in senescent cells. 6. These results suggest that the unresponsiveness of senescent fibroblasts to PDGF stimulation may be due to increased levels of PTRF and the formation of caveolae, which, in turn sequester growth receptors, such as PDGFR and its signalling molecules.

Met1-linked ubiquitination in cell signaling regulation.

Met1-linked ubiquitination (Met1-Ub), also known as linear ubiquitination, is a newly identified atypical type of polyubiquitination that is assembled via the N-terminal methionine (Met1) rather than an internal lysine (Lys) residue of ubiquitin. The linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L and SHARPIN is the sole E3 ubiquitin ligase that specifically generates Met1-linked ubiquitin chains. The physiological role of LUBAC-mediated Met1-Ub has been first described as activating NF-κB signaling through the Met1-Ub modification of NEMO. However, accumulating evidence shows that Met1-Ub is broadly involved in other cellular pathways including MAPK, Wnt/ß-Catenin, PI3K/AKT and interferon signaling, and participates in various cellular processes including angiogenesis, protein quality control and autophagy, suggesting that Met1-Ub harbors a potent signaling capacity. Here, we review the formation and cellular functions of Met1-linked ubiquitin chains, with an emphasis on the recent advances in the cellular mechanisms by which Met1-Ub controls signaling transduction.

Serine/threonine-protein phosphatase 2A physically interacts with human telomerase reverse transcriptase hTERT and regulates its subcellular distribution.

Telomerase plays fundamental roles in bypassing cellular aging and promoting cancer progression by maintaining telomere homeostasis and telomere-independent activities. However, the molecular mechanisms by which telomerase provokes aging and cancer are far from being fully understood. In a search for proteins interacting with human telomerase reverse transcriptase hTERT by the yeast two-hybrid screen using hTERT T-motif as bait, we identified PP2A scaffolding subunit PR65 alpha isoform as an hTERT interacting partner. We showed that both PP2A catalytic subunit PP2AC and scaffolding subunit PR65 interacted with hTERT in vivo and in vitro and inhibited telomerase activity. In addition, we found that PP2A prevented the interaction of hTERT with 14-3-3θ signaling protein, an hTERT binding partner that is required for nuclear localization of hTERT. Activation of PP2A by overexpression of PP2AC or PR65 led to cytoplasmic accumulation of hTERT, which was reversed by treatment with PP2A inhibitor okadaic acid. Together, these observations suggest that PP2A regulates hTERT subcellular localization, in addition to its inhibitory effects on telomerase activity.

Randomized trial of transcutaneous auricular vagus nerve stimulation on patients with disorders of consciousness: A study protocol.

Background: Transcutaneous auricular vagus nerve stimulation (taVNS) has recently been explored for the treatment of Disorders of consciousness (DoC) caused by traumatic brain injury. The evidence of taVNS during the consciousness recovery has been recently reported. However, the mechanism of taVNS in the recovery of consciousness is not clear. This study attempts to investigate the effectiveness of taVNS in DoC by means of Coma Recovery Scale-Revised (CRS-R), Magnetic resonance imaging (MRI), Electrophysiology (EEG), and Single-molecular array (Simoa). Methods/design: Nighty patients with DoC acquired brain injury are randomized into one of three groups receiving sham taVNS or active taVNS (just left and left or right), respectively. Each of the three groups will experience a 40 days cycle (every 10 days for a small period, baseline 2 weeks, intervention 2 weeks, 40 min per day, 5 days per week, then no intervention for 2 weeks, intervention 2 weeks, 40 min per day, and 5 days per week). Primary outcomes (CRS-R) will be recorded five times during every period. Secondary outcomes will be recorded at the first and at the last period [MRI, EEG, Phosphorylated tau (P-tau), and Neurofilament light chain (NFL)]. We will take notes the adverse events and untoward effects during all cycles. Discussion: Transcutaneous auricular vagus nerve stimulation as a painless, non-invasive, easily applied, and effective therapy was applied for treatment of patients with depression and epilepsy several decades ago. Recent progress showed that taVNS has behavioral effects in the consciousness recovery. However, there is no clinical evidence to support the effects of taVNS on brain activity. Therefore, we will design a randomized controlled trial to evaluate the effectiveness and safety of taVNS therapy for DoC, and explore neural anatomy correlated to taVNS during the consciousness recovery. Finally, this protocol also tests some biomarkers along with the recovery of consciousness. Clinical Trial Registration: Chinese Clinical Trial Registry, ChiCTR2100045161. Registered on 9 April 2021.

Down-regulation of the cavin family proteins in breast cancer.

Caveolae are abundant membrane domain on the cell surface of many mammalian cell types and are implicated in a wide range of physiological processes. The caveolae structural protein caveolin-1 is often mutated or deregulated in cancer, and cavin family protein serum deprivation response factor-related gene product that binds to C-kinase (SRBC) has been found to be epigenetically inactivated in lung, breast, and gastric cancer. Both caveolin-1 and SRBC have been proposed to function as tumor suppressors. Polymerase 1 and transcript release factor (PTRF) is the essential component for caveolae formation. The regulation of PTRF expression in cancer has not been characterized. We report here that the cavin family protein PTRF, SRBC and serum deprivation response protein were down regulated in breast cancer cell lines and breast tumor tissue. We further show that down-regulation of PTRF in breast cancer cells was associated with the promoter methylation. As caveolin-1 and cavin family proteins are required for caveolae formation and function, the reported tumor suppression function of caveolin-1 and SRBC may be due to the deregulation of caveolae and its down-stream signaling. Thus, the caveolae is a potential therapeutic target and the expression of cavin family proteins could be a useful prognostic indicator of breast cancer progression.

Optimized protocol to detect protein UFMylation in cells and in vitro via immunoblotting.

Ubiquitin-fold modifier 1 (UFM1) system is a recently identified ubiquitin-like modification with essential biological functions. Similar to ubiquitination, the covalent conjugation of UFM1 (UFMylation) to target proteins involves a three-step enzymatic cascade catalyzed sequentially by UFM1-activating enzyme 5 (UBA5, E1), UFM1-conjugating enzyme 1 (UFC1, E2), and UFM1-specific ligase 1 (UFL1, E3). Here, we provide an optimized protocol adapted to previously reported methods for detecting the UFMylation of target protein in human cells and in vitro assays, respectively, with high reliability and reproducibility. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).

Ufl1 deficiency causes skin pigmentation by up-regulation of Endothelin-1.

Ufmylation (UFM1 modification) is a newly identified ubiquitin-like modification system involved in numerous cellular processes. However, the regulatory mechanisms and biological functions of this modification remain mostly unknown. We have recently reported that Ufmylation family genes have frequent somatic copy number alterations in human cancer including melanoma, suggesting involvement of Ufmylation in skin function and disease. UFL1 is the only known Ufmylation E3-like ligase. In this study, we generated the skin-specific Ufl1 knockout mice and show that ablation of Ufl1 caused epidermal thickening, pigmentation and shortened life span. RNA-Seq analysis indicated that Ufl1 deletion resulted in upregulation of the genes involved in melanin biosynthesis. Mechanistically, we found that Endothelin-1 (ET-1) is a novel substrate of Ufmylation and this modification regulates ET-1 stability, and thereby deletion of Ufl1 upregulates the expression and secretion of ET-1, which in turn results in up-regulation of genes in melanin biosynthesis and skin pigmentation. Our findings establish the role of Ufl1 in skin pigmentation through Ufmylation modification of ET-1 and provide opportunities for therapeutic intervention of skin diseases.

Differential expression of oncogenic miRNAs in proliferating and senescent human fibroblasts.

miRNAs are a class of non-coding RNAs that play fundamental roles through the post-transcriptional regulation of target mRNAs. miRNAs have been shown to regulate a broad spectrum of biological activities, including development, differentiation, cell death, and oncogenesis. However, little is known about their contribution to cellular senescence. The authors analyzed the expression of 576 miRNAs in proliferating and senescent normal human fibroblasts by microarray, and identified 12 miRNAs that were differentially expressed in proliferating and senescent fibroblasts. Interestingly, all six miRNAs that were down-regulated in senescent cells had been previously reported to be aberrantly expressed in tumor cells. It was further showed that inhibition of miR-17-5p and miR-20a by 2'-O-methyl antisense oligoribonucleotides resulted in the induction of senescent phenotypes in WI-38 cells.

Telomere Dysfunction in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is an age-dependent progressive and fatal lung disease of unknown etiology, which is characterized by the excessive accumulation of extracellular matrix inside the interstitial layer of the lung parenchyma that leads to abnormal scar architecture and compromised lung function capacity. Recent genetic studies have attributed the pathological genes or genetic mutations associated with familial idiopathic pulmonary fibrosis (IPF) and sporadic IPF to telomere-related components, suggesting that telomere dysfunction is an important determinant of this disease. In this study, we summarized recent advances in our understanding of how telomere dysfunction drives IPF genesis. We highlighted the key role of alveolar stem cell dysfunction caused by telomere shortening or telomere uncapping, which bridged the gap between telomere abnormalities and fibrotic lung pathology. We emphasized that senescence-associated secretory phenotypes, innate immune cell infiltration, and/or inflammation downstream of lung stem cell dysfunction influenced the native microenvironment and local cell signals, including increased transforming growth factor-beta (TGF-ß) signaling in the lung, to induce pro-fibrotic conditions. In addition, the failed regeneration of new alveoli due to alveolar stem cell dysfunction might expose lung cells to elevated mechanical tension, which could activate the TGF-ß signaling loop to promote the fibrotic process, especially in a periphery-to-center pattern as seen in IPF patients. Understanding the telomere-related molecular and pathophysiological mechanisms of IPF would provide new insights into IPF etiology and therapeutic strategies for this fatal disease.