Identification of cytotoxic T cells and their T cell receptor sequences targeting COVID-19 using MHC class I-binding peptides.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) was first reported in China in December 2019, various variants have been identified in different areas of the world such as United Kingdom (alpha), South Africa (beta and omicron), Brazil (gamma), and India (delta). Some of SARS-CoV-2 variants, each of which is characterized by a unique mutation(s) in spike protein, are concerned due to their high infectivity and the capability to escape from neutralizing antibodies elicited by vaccinations. To identify peptide epitopes that are derived from SARS-CoV-2 viral proteins and possibly induce CD8+ T cell immunity, we investigated SARS-CoV-2-derived peptides that are likely to bind to major histocompatibility complex (MHC) class I molecules. We identified a total of 15 peptides that bind to human leukocyte antigen (HLA)-A*24:02, HLA-A*02:01, or HLA-A*02:06, and possibly induce cytotoxic T lymphocytes (CTLs); thirteen of them corresponded to ORF1ab polyprotein, one peptide to spike protein and the remaining one to membrane glycoprotein. CD8+ T cells that recognize these peptides were detected in peripheral blood samples in three individuals recovered from COVID-19 as well as non-infected individuals. Since most of these peptides are commonly conserved among other coronaviruses including SARS-CoV and/or MERS-CoV, these might be useful to maintain T cell responses to coronaviruses that are pandemic at present and will become the future threat. We could define pairs of TRA and TRB sequences of nine CTL clones that recognize SARS-CoV-2-derived peptides. We might use these SARS-CoV-2-derived peptide-reactive TCR sequences for investigating the history of SARS-CoV-2 infection.

Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems.

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Selective and easy detection of the Porphyromonas gingivalis fimA type II and IV genes by loop-mediated isothermal amplification.

Porphyromonas gingivalis fimbrillin (fimA) type II and IV, the definitive factors for periodontitis, are also found to be associated with systemic diseases. To detect the fimA type II and IV genes easily and rapidly, we used the loop-mediated isothermal amplification (LAMP) method. The LAMP method showed high specificity as DNA from the P. gingivalis HW24D1 strain could only be amplified by the type II-specific primers and that from the W83 strain could only be amplified by the type IV-specific primers. Pathogens, namely, Streptococcus sobrinus, S. mutans, and Candida species, lack the type II and IV genes, and hence, were not detected by the LAMP reaction. Both bacterial cells and purified DNA could be used in the LAMP reaction. The LAMP reaction was highly sensitive and both type II and type IV genes could be detected in 1000 DNA molecules. In the bacterial suspensions of HW24D1 and W83 strains, type II and type IV genes, respectively, could be detected in 100 bacterial cells. We examined the type II and type IV genes in the dental plaques from 22 P. gingivalis-positive patients using the LAMP method. Only one person was found to be positive for the type II gene (4.5%). For the type IV gene, 3 positive cases (13.6%) were identified. Moreover, type II and type IV genes could be detected simultaneously using a multiplex amplification primer of fimA type II and type IV, under visible light. Thus, we established a selective and easy method to detect P. gingivalis fimA type II and IV genes using LAMP.

Depletion of Csk preferentially reduces the protein level of LynA in a Cbl-dependent manner in cancer cells.

There are eight human Src-family tyrosine kinases (SFKs). SFK members c-Src, c-Yes, Fyn, and Lyn are expressed in various cancer cells. SFK kinase activity is negatively regulated by Csk tyrosine kinase. Reduced activity of Csk causes aberrant activation of SFKs, which can be degraded by a compensatory mechanism depending on Cbl-family ubiquitin ligases. We herein investigated whether all SFK members are similarly downregulated by Cbl-family ubiquitin ligases in cancer cells lacking Csk activity. We performed Western blotting of multiple cancer cells knocked down for Csk and found that the protein levels of the 56 kDa isoform of Lyn (LynA), 53 kDa isoform of Lyn (LynB), c-Src, and Fyn, but not of c-Yes, were reduced by Csk depletion. Induction of c-Cbl protein levels was also observed in Csk-depleted cells. The reduction of LynA accompanying the depletion of Csk was significantly reversed by the knockdown for Cbls, whereas such significant recovery of LynB, c-Src, and Fyn was not observed. These results suggested that LynA is selectively downregulated by Cbls in cancer cells lacking Csk activity.

The histone methyltransferase Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) is involved in human carcinogenesis.

Histone lysine methylation plays a fundamental role in chromatin organization. Although a set of histone methyltransferases have been identified and biochemically characterized, the pathological roles of their dysfunction in human cancers are still not well understood. In this study, we demonstrate important roles of WHSC1L1 in human carcinogenesis. Expression levels of WHSC1L1 transcript were significantly elevated in various human cancers including bladder carcinoma. Immunohistochemical analysis of bladder, lung, and liver cancers confirmed overexpression of WHSC1L1. WHSC1L1-specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of bladder and lung cancer cell lines. WHSC1L1 knockdown induced cell cycle arrest at the G(2)/M phase followed by multinucleation of cancer cells. Expression profile analysis using Affymetrix GeneChip(®) showed that WHSC1L1 affected the expression of a number of genes including CCNG1 and NEK7, which are known to play crucial roles in the cell cycle progression at mitosis. As WHSC1L1 expression is significantly low in various normal tissues including vital organs, WHSC1L1 could be a good candidate molecule for development of novel treatment for various types of cancer.

RB1 methylation by SMYD2 enhances cell cycle progression through an increase of RB1 phosphorylation.

It is well known that RB functions are regulated by posttranslational modifications such as phosphorylation and acetylation, but the significance of lysine methylation on RB has not been fully elucidated. Our expression analysis of SMYD2 by quantitative real-time polymerase chain reaction showed that expression levels of SMYD2 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (P < .0001), and its expression levels in tumor tissues were much higher than those of any other normal tissues. SMYD2 knockdown resulted in the suppression of cancer cell growth, and cell cycle analysis indicated that SMYD2 might play a crucial role in the G(1)/S transition. According to an in vitro methyltransferase assay, we found that SMYD2 methylates RB1 protein, and liquid chromatography-tandem mass spectrometry analysis revealed lysine 810 of RB1 to be methylated by SMYD2. Importantly, this methylation enhanced Ser 807/811 phosphorylation of RB1 both in vitro and in vivo. Furthermore, we demonstrated that methylated RB1 accelerates E2F transcriptional activity and promotes cell cycle progression. SMYD2 is an important oncoprotein in various types of cancer, and SMYD2-dependent RB1 methylation at lysine 810 promotes cell cycle progression of cancer cells. Further study may explore SMYD2-dependent RB1 methylation as a potential therapeutic target in human cancer.

Histone lysine methyltransferase SETD8 promotes carcinogenesis by deregulating PCNA expression.

Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysine methylation. We found that SETD8 methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNA and the flap endonuclease FEN1. Loss of PCNA methylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNA mutant were more susceptible to DNA damage. An increase of methylated PCNA was found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis.

The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene.

A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogenesis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real-time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di-methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down-regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G(1) arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition.

Histone lysine methyltransferase Wolf-Hirschhorn syndrome candidate 1 is involved in human carcinogenesis through regulation of the Wnt pathway.

A number of histone methyltransferases have been identified and biochemically characterized, but the pathologic roles of their dysfunction in human diseases like cancer are not well understood. Here, we demonstrate that Wolf-Hirschhorn syndrome candidate 1 (WHSC1) plays important roles in human carcinogenesis. Transcriptional levels of this gene are significantly elevated in various types of cancer including bladder and lung cancers. Immunohistochemical analysis using a number of clinical tissues confirmed significant up-regulation of WHSC1 expression in bladder and lung cancer cells at the protein level. Treatment of cancer cell lines with small interfering RNA targeting WHSC1 significantly knocked down its expression and resulted in the suppression of proliferation. Cell cycle analysis by flow cytometry indicated that knockdown of WHSC1 decreased the cell population of cancer cells at the S phase while increasing that at the G(2)/M phase. WHSC1 interacts with some proteins related to the WNT pathway including ß-catenin and transcriptionally regulates CCND1, the target gene of the ß-catenin/Tcf-4 complex, through histone H3 at lysine 36 trimethylation. This is a novel mechanism for WNT pathway dysregulation in human carcinogenesis, mediated by the epigenetic regulation of histone H3. Because expression levels of WHSC1 are significantly low in most normal tissue types, it should be feasible to develop specific and selective inhibitors targeting the enzyme as antitumor agents that have a minimal risk of adverse reaction.

Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker.

The emphasis in anticancer drug discovery has always been on finding a drug with great antitumor potential but few side-effects. This can be achieved if the drug is specific for a molecular site found only in tumor cells. Here, we find the enhancer of zeste homolog 2 (EZH2) to be highly overexpressed in lung and other cancers, and show that EZH2 is integral to proliferation in cancer cells. Quantitative real-time PCR analysis revealed higher expression of EZH2 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpress EZH2, using cDNA microarray analysis. Immunohistochemical analysis showed positive staining for EZH2 in 14 of 29 cases of bladder cancer, 135 of 292 cases of non-small-cell lung cancer (NSCLC), and 214 of 245 cases of colorectal cancer, whereas no significant staining was observed in various normal tissues. We found elevated expression of EZH2 to be associated with poor prognosis for patients with NSCLC (P = 0.0239). In lung and bladder cancer cells overexpressing EZH2, suppression of EZH2 using specific siRNAs inhibited incorporation of BrdU and resulted in significant suppression of cell growth, even though no significant effect was observed in the normal cell strain CCD-18Co, which has undetectable EZH2. Because EZH2 expression was scarcely detectable in all normal tissues we examined, EZH2 shows promise as a tumor-specific therapeutic target. Furthermore, as elevated levels of EZH2 are associated with poor prognosis of patients with NSCLC, its overexpression in resected specimens could prove a useful molecular marker, indicating the necessity for a more extensive follow-up in some lung cancer patients after surgical treatment.