Securin acetylation prevents precocious separase activation and premature sister chromatid separation.

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

SPTBN1 Mediates the Cytoplasmic Constraint of PTTG1, Impairing Its Oncogenic Activity in Human Seminoma.

Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 (MMP-2) and E-cadherin (CDH1). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma.

CENP-F-dependent DRP1 function regulates APC/C activity during oocyte meiosis I.

Chromosome segregation is initiated by cohesin degradation, which is driven by anaphase-promoting complex/cyclosome (APC/C). Chromosome cohesin is removed by activated separase, with the degradation of securin and cyclinB1. Dynamin-related protein 1 (DRP1), a component of the mitochondrial fission machinery, is related to cyclin dynamics in mitosis progression. Here, we show that DRP1 is recruited to the kinetochore by centromeric Centromere protein F (CENP-F) after nuclear envelope breakdown in mouse oocytes. Loss of DRP1 during prometaphase leads to premature cohesin degradation and chromosome segregation. Importantly, acute DRP1 depletion activates separase by initiating cyclinB1 and securin degradation during the metaphase-to-anaphase transition. Finally, we demonstrate that DRP1 is bound to APC2 to restrain the E3 ligase activity of APC/C. In conclusion, DRP1 is a CENP-F-dependent atypical spindle assembly checkpoint (SAC) protein that modulates metaphase-to-anaphase transition by controlling APC/C activity during meiosis I in oocytes.

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Genome-wide analysis of DNaseI hypersensitivity unveils open chromatin associated with histone H3 modifications after areca nut with lime exposure.

Research over the years revealed that precocious anaphase, securin overexpression, and genome instability in both target and nontarget cells are significantly associated with the increased risk of areca nut (AN) and lime-induced oral, esophageal, and gastric cancers. Further, hyperphosphorylation of Rb and histone H3 epigenetic modifications both globally and in the promoter region of the securin gene were demonstrated after AN + lime exposure. This study aims whether the extract of raw AN + lime relaxes chromatin structure which further facilitates the histone H3 epigenetic modifications during the initial phase of carcinogenesis. Three groups of mice (10 in each group) were used. The treated group consumed 1 mg/day/mice of AN extract with lime ad libitum in the drinking water for 60 days. The dose was increased by 1 mg every 60 days. Isolated nuclei were digested with DNaseI and 2 kb and below DNA was eluted from the agarose gel, purified and PCR amplified by using securin and GAPDH primers. Securin and E2F1 expression, pRb phosphorylation, and histone epigenetic modifications were analyzed by immunohistochemistry. The number of DNA fragments within 2 kb in size after DNaseI treatment was higher significantly in AN + lime exposed tissue samples than in the untreated one. The PCR result showed that the number of fragments bearing securin gene promoter and GAPDH gene was significantly higher in AN + lime exposed DNaseI-treated samples. Immunohistochemistry data revealed increased Rb hyperphosphorylation, upregulation of E2F1, and securin in the AN + lime-treated samples. Increased trimethylation of histone H3 lysine 4 and acetylation of H3 lysine 9 and 18 were observed globally in the treated samples. Therefore, the results of this study have led to the hypothesis that AN + lime exposure relaxes the chromatin, changes the epigenetic landscape, and deregulates the Rb-E2F1 circuit which might be involved in the upregulation of securin and some other proto-oncogenes that might play an important role in the initial phases of AN + lime mediated carcinogenesis.

Impaired Cdc20 signaling promotes senescence in normal cells and apoptosis in non-small cell lung cancer cells.

Cellular senescence is a form of irreversible growth arrest that cancer cells evade. The cell division cycle protein 20 homolog (Cdc20) is a positive regulator of cell division, but how its dysregulation may relate to senescence is unclear. Here, we find that Cdc20 mRNA and protein expression are downregulated in stress-induced premature senescent lung fibroblasts in a p53-dependent manner. Either Cdc20 downregulation or inhibition of anaphase-promoting complex/cyclosome (APC/C) is sufficient to induce premature senescence in lung fibroblasts, while APC/C activation inhibits stress-induced premature senescence. Mechanistically, we show both Cdc20 downregulation and APC/C inhibition induce premature senescence through glycogen synthase kinase (GSK)-3ß-mediated phosphorylation and downregulation of securin expression. Interestingly, we determined Cdc20 expression is upregulated in human lung adenocarcinoma. We find that downregulation of Cdc20 in non-small cell lung cancer (NSCLC) cells is sufficient to inhibit cell proliferation and growth in soft agar and to promote apoptosis, but not senescence, in a manner dependent on downregulation of securin following GSK-3ß-mediated securin phosphorylation. Similarly, we demonstrate securin expression is downregulated and cell viability is inhibited in NSCLC cells following inhibition of APC/C. Furthermore, we show chemotherapeutic drugs downregulate both Cdc20 and securin protein expression in NSCLC cells. Either Cdc20 downregulation by siRNA or APC/C inhibition sensitize, while securin overexpression inhibits, chemotherapeutic drug-induced NSCLC cell death. Together, our findings provide evidence that Cdc20/APC/C/securin-dependent signaling is a key regulator of cell survival, and its disruption promotes premature senescence in normal lung cells and induces apoptosis in lung cancer cells that have bypassed the senescence barrier.

Identification of a novel substrate motif of yeast separase and deciphering the recognition specificity using AlphaFold2 and molecular dynamics simulation.

Separase is a giant cysteine protease and has multiple crucial functions. The most well-known substrate of separase is the kleisin subunit of cohesin, the cleavage of which triggers chromosome segregation during cell division (Uhlmann et al., 1999; Kamenz and Hauf, 2016) [1,2]. Recently, separase has also been found to cleave MCL-1 or BCL-XL proteins to trigger apoptosis (Hellmuth and Stemmann, 2020) [3]. Although substrate recognition through a short sequence right upstream of the cleavage site is well established, recent studies suggested that sequence elements outside this minimum cleavage site are required for optimal cleavage activity and specificity (Rosen et al., 2019; Uhlmann et al., 2000) [4,5]. However, the sequences and their underlying mechanism are largely unknown. To further explore the substrate determinants and recognition mechanism, we carried out sequence alignments and found a conserved motif downstream of the cleavage site in budding yeast. Using Alphafold2 and molecular dynamics simulations, we found this motif is recognized by separase in a conserved cleft near the binding groove of its inhibitor securin. Their binding is mutually exclusive and requires conformation changes of separase. These findings provide deeper insights into substrate recognition and activation of separase, and paved the way for discovering more substrates of separase.

Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase.

Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and can cooperate with the proteasome for their degradation. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase, resulting in co-degradation of separase along with securin. If true, Cdc48 would be important in mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

The pituitary tumour-transforming gene 1/delta-like homologue 1 pathway plays a key role in liver fibrogenesis.

BACKGROUND AND AIMS: PTTG1 is almost undetectable in adult livers but is highly expressed in hepatocarcinoma. While little is known about its involvement in liver fibrosis, PTTG1 expression is associated with DLK1. We assessed the role of the PTTG1/DLK1 pathway in fibrosis progression and the potential therapeutic effect of PTTG1 silencing in fibrosis. METHODS: Pttg1 and Dlk1 were studied in liver and isolated cell populations of control and fibrotic rats and in human liver biopsies. The fibrotic molecular signature was analysed in Pttg1-/- and Pttg1+/+ fibrotic mice. Finally, Pttg1 silencing was evaluated in rats as a novel antifibrotic therapy. RESULTS: Pttg1 and Dlk1 mRNA selectively increased in fibrotic rats paralleling fibrosis progression. Serum DLK1 concentrations correlated with hepatic collagen content and systemic and portal haemodynamics. Human cirrhotic livers showed greater PTTG1 and DLK1 transcript abundance than non-cirrhotic, and reduced collagen was observed in Pttg1 Pttg1-/- mice. The liver fibrotic molecular signature revealed lower expression of genes related to extracellular matrix remodelling including Mmp8 and 9 and Timp4 and greater eotaxin and Mmp13 than fibrotic Pttg1+/+ mice. Finally, interfering Pttg1 resulted in reduced liver fibrotic area, lower α-Sma and decreased portal pressure than fibrotic animals. Furthermore, Pttg1 silencing decreased the transcription of Dlk1, collagens I and III, Pdgfrß, Tgfrß, Timp1, Timp2 and Mmp2. CONCLUSIONS: Pttg1/Dlk1 are selectively overexpressed in the cirrhotic liver and participate in ECM turnover regulation. Pttg1 disruption decreases Dlk1 transcription and attenuates collagen deposition. PTTG1/DLK1 signalling is a novel pathway for targeting the progression of liver fibrosis.

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast.

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

SP1-mediated transcriptional activation of PTTG1 regulates the migration and phenotypic switching of aortic vascular smooth muscle cells in aortic dissection through MAPK signaling.

Pituitary tumor-transforming gene 1 (PTTG1) has been found to be associated with the process of cell proliferation and invasion, and is highly expressed in aortic dissection (AD). However, its potential role and underlying mechanism in AD remain uncertain. This study aims at elucidating the roles of specificity protein 1 (SP1) and PTTG1 in the migration and phenotypic switching of aortic vascular smooth muscle cells (VSMCs) in AD. Aortic samples were collected from 35 patients with AD for examination of PTTG1 expression in the tissues by qPCR, western blot and immunofluorescence. Human aortic vascular smooth muscle cells (HAVSMCs) were stimulated with platelet-derived growth factor-BB (PDGF-BB) to establish the cellular model of AD. PTTG1 expression in VSMCs was also examined by qPCR and western blot. Cell viability was detected by CCK-8, cell proliferation by EdU staining and cell migration by wound healing and transwell. Western blot was then performed to assay migration-related proteins. After interference with PTTG1, the levels of smooth muscle pthenotypic switch markers smooth muscle protein 22 alpha (SM22-α) and osteopontin (OPN) were detected by qPCR, western blot and immunofluorescence. The binding of SP1 and PTTG1 was verified with dual-luciferase reporter assay and chromatin immunoprecipitation assay (ChIP). PTTG1 overexpression was found in AD patients. Interference with PTTG1 attenuated the proliferation and migration of PDGF-BB-stimulated HAVSMCs, in addition to their switching from contractile phenotype to synthetic phenotype. Transcription factor SP1 was up-regulated in PDGF-BB-stimulated HAVSMCs, combined with PTTG1 promoter sequence and regulated PTTG1 expression, whose overexpression reversed the effects of PTTG1 interference on cell proliferation, migration and phenotypic switching. SP1 transcriptional activation of PTTG1 activated MAPK/ERK signaling pathway. In conclusion, SP1 transcriptional activation of PTTG1 regulates the migration and phenotypic transformation of HAVSMCs in AD by MAPK Signaling.

Identification of disease-relevant modulators of the SHH pathway in the developing brain.

Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.

A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes.

Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.

Structural basis of human separase regulation by securin and CDK1-cyclin B1.

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.

Real-Time Monitoring of APC /C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts.

The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C-Cdk1 activating the APC/C and the APC/C inactivating Cdk1-constitutes the core of the biochemical cell cycle oscillator.Since its discovery three decades ago, the mechanisms of APC /C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC /C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC /C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC /C activity in Xenopus laevis egg extracts using APC /C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC /C mutants or investigate other mechanisms of APC /C regulation.

Checkpoint kinases are required for oocyte meiotic progression by the maintenance of normal spindle structure and chromosome condensation.

Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.

PTTG1 knockdown enhances radiation-induced antitumour immunity in lung adenocarcinoma.

AIM: Ionizing radiation (IR) can induce local and systemic antitumour immune responses to some degree and augment immunotherapeutic efficacy. IR may also increase residual tumour cell invasion and elicit immunosuppression in the tumour microenvironment (TME). It remains poorly understand, whether IR leads to immune negative response or invasive capacity increases in lung adenocarcinoma (LAC). MATERIALS AND METHODS: RNA interference (RNAi) was used to silence pituitary tumour-transforming gene-1 (PTTG1) and SMAD3 expression in LAC cells. A coculture system of tumour cells and PBMCs was constructed. Cells were exposed to different doses (0, 4 and 8 Gy) of X-ray irradiation. Flow cytometric analysis and Transwell assays were applied. An orthotopic Lewis lung cancer (LLC) mouse tumour model was established. Bioluminescence imaging (BLI) was used. LLC tumours were exposed to a single 15 Gy dose of X-ray irradiation. KEY FINDINGS: PTTG1 knockdown reinforced the inhibitory effect of IR on the invasive ability of A549 cells and enhanced the antitumour T cell immunity induced by IR via the transforming growth factor-ß1 (TGF-ß1)/SMAD3 pathway. Positive antitumour immune response and immunosuppression were simultaneously triggered by a single 15 Gy dose of local tumour irradiation. PTTG1 knockdown weakened invasive capacity and promoted the immune response balance induced by IR to tilt towards active immunity, which contributed to reduce metastasis and prolonged overall survival (OS) in orthotopic LLC tumour-bearing mouse. SIGNIFICANCE: Targeted blockade of PTTG1 and the TGF-ß1/SMAD3 pathway may ameliorate the immunosuppressive TME and enhance the systemic antitumour immune response induced by a single high-dose IR treatment.

Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases.

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

Alteration of Pituitary Tumor Transforming Gene 1 by MicroRNA-186 and 655 Regulates Invasion Ability of Human Oral Squamous Cell Carcinoma.

BACKGROUND: Pituitary tumor-transforming gene 1 (PTTG1) was recently shown to be involved in the progression as well as the metastasis of cancers. However, their expression and function in the invasion of oral squamous cell carcinoma (SCC) remain unclear. METHODS: The expressions of PTTG1 and PTTG1-targeted miRNA in oral SCC cell lines and their invasion capability depended on PTTG1 expression were analyzed by quantitative RT-PCR, Western blots, the transwell insert system and Zymography. RESULTS: Invasion abilities were decreased in oral SCC cells treated with siRNA-PTTG1. When PTTG1 were downregulated in oral SCC cells treated with microRNA-186 and -655 inhibited their invasion abilities via MMP-9 activity. CONCLUSIONS: These results indicate that alteration of expression of PTTG1 in oral SCC cells by newly identified microRNA-186 and -655 can regulate invasion activity. Therefore, these data offer new insights into further understanding PTTG1 function in oral SCC and should provide new strategies for diagnostic markers for oral SCC.

Structure and Function of the Separase-Securin Complex.

Separase is a large cysteine protease in eukaryotes and has crucial roles in many cellular processes, especially chromosome segregation during mitosis and meiosis, apoptosis, DNA damage repair, centrosome disengagement and duplication, spindle stabilization and elongation. It dissolves the cohesion between sister chromatids by cleaving one of the subunits of the cohesin ring for chromosome segregation. The activity of separase is tightly controlled at many levels, through direct binding of inhibitory proteins as well as posttranslational modification. Dysregulation of separase activity is linked to cancer and genome instability, making it a target for drug discovery. One of the best-known inhibitors of separase is securin, which has been identified in yeast, plants, and animals. Securin forms a tight complex with separase and potently inhibits its catalytic activity. Recent structures of the separase-securin complex have revealed the molecular mechanism for the inhibitory activity of securin. A segment of securin is bound in the active site of separase, thereby blocking substrate binding. Securin itself is not cleaved by separase as its binding mode is not compatible with catalysis. Securin also has extensive interactions with separase outside the active site, consistent with its function as a chaperone to stabilize this enzyme.