An immune gene signature to predict prognosis and immunotherapeutic response in lung adenocarcinoma.

Lung adenocarcinoma is one of the most common malignant tumors worldwide. The purpose of this study was to construct a stable immune gene signature for prediction of prognosis (IGSPP) and response to immune checkpoint inhibitors (ICIs) therapy in LUAD patients. Five genes were screened by weighted gene coexpression network analysis, Cox regression and LASSO regression analyses and were used to construct the IGSPP. The survival rate of the IGSPP low-risk group was higher than that of the IGSPP high-risk group. Multivariate Cox regression analysis showed that IGSPP could be used as an independent prognostic factor for the overall survival of LUAD patients. IGSPP genes were enriched in cell cycle pathways. IGSPP gene mutation rates were higher in the high-risk group. CD4 memory-activated T cells, M0 and M1 macrophages had higher infiltration abundance in the high-risk group, which was associated with poor overall survival. In contrast, the abundance of resting CD4 memory T cells, monocytes, resting dendritic cells and resting mast cells associated with a better prognosis was higher in the low-risk group. TIDE scores and the expressions of different immune checkpoints showed that patients in the high-risk IGSPP group benefited more from ICIs treatment. In short, an IGSPP of LUAD was constructed and characterized. It could be used to predict the prognosis and benefits of ICIs treatment in LUAD patients.

PRKCDBP Methylation is a Potential and Promising Candidate Biomarker for Non-small Cell Lung Cancer.

BACKGROUND: The occurrence and development of lung cancer are closely linked to epigenetic modification. Abnormal DNA methylation in the CpG island region of genes has been found in many cancers. Protein kinase C delta binding protein (PRKCDBP) is a potential tumor suppressor and its epigenetic changes are found in many human malignancies. This study investigated the possibility of PRKCDBP methylation as a potential biomarker for non-small cell lung cancer (NSCLC). METHODS: We measured the methylation levels of PRKCDBP in the three groups of NSCLC tissues. Promoter activity was measured by the dual luciferase assay, with 5'-aza-deoxycytidine to examine the effect of demethylation on the expression level of PRKCDBP. RESULTS: The methylation levels of PRKCDBP in tumor tissues and 3 cm para-tumor were higher than those of distant (>10 cm) non-tumor tissues. Receiver operating characteristic (ROC) curve analysis between tumor tissues and distant non-tumor tissues showed that the area under the line (AUC) was 0.717. Dual luciferase experiment confirmed that the promoter region was able to promote gene expression. Meanwhile, in vitro methylation of the fragment (PRKCDBP_Me) could significantly reduce the promoter activity of the fragment. Demethylation of 5'-aza-deoxycytidine in lung cancer cell lines A549 and H1299 showed a significant up-regulation of PRKCDBP mRNA levels. CONCLUSIONS: PRKCDBP methylation is a potential and promising candidate biomarker for non-small cell lung cancer.

The deubiquitinating enzyme USP20 regulates the stability of the MCL1 protein.

Chemoresistance is a major obstacle faced by oesophageal cancer patients and is synonymous with a poor prognosis. MCL1 is a pivotal member of the anti-apoptotic Bcl-2 protein family, which has been found to play an important role in cell survival, proliferation, differentiation and chemoresistance. Thus, it might be an ideal target for treating oesophageal cancer patients. Although it is known that MCL1 is degraded via the ubiquitin-proteasome system, the deubiquitylating enzyme (DUB) responsible for stabilizing MCL1 remains elusive to date. Herein, we demonstrate that Ubiquitin-Specific Protease 20 (USP20) is a novel regulator of the apoptotic signaling pathway. Moreover, USP20 could regulate the deubiquitination of MCL1 to, in turn, regulate its stability. Increased expression of USP20 was correlated with increased levels of MCL1 protein in human patient samples. In addition, depletion of USP20 could increase the polyubiquitination of MCL1, thereby increasing the conversion rate of MCL1 and the sensitivity of cells to chemotherapy. Overall, our findings indicate that the USP20-MCL1 axis might play a key role in the apoptotic signaling pathway.

Regulation of survivin protein stability by USP35 is evolutionarily conserved.

Survivin is the key component of the chromosomal passenger complex and plays important roles in the regulation of cell division. Survivin has also been implicated in the regulation of apoptosis and tumorigenesis. Although the survivin protein has been reported to be degraded by a ubiquitin/proteasome-dependent mechanism, whether there is a DUB that is involved in the regulation of its protein stability is largely unknown. Using an expression library containing 68 deubiquitinating enzymes, we found that ubiquitin-specific-processing protease 35 (USP35) regulates survivin protein stability in an enzymatic activity-dependent manner. USP35 interacted with and promoted the deubiquitination of the survivin protein. USP38, an ortholog of USP35 encoded by the human genome, is also able to regulate survivin protein stability. Moreover, we found that the deubiquitinating enzyme DUBAI, the Drosophila homolog of human USP35, is able to regulate the protein stability of Deterin, the Drosophila homolog of survivin. Interestingly, USP35 also regulated the protein stability of Aurora B and Borealin which are also the component of the chromosomal passenger complex. By regulating protein stabilities of chromosomal passenger complex components, USP35 regulated cancer cell proliferation. Taken together, our work uncovered an evolutionarily conserved relationship between USP35 and survivin that might play an important role in cell proliferation.

Self-stabilizing regulation of deubiquitinating enzymes in an enzymatic activity-dependent manner.

Deubiquitinating enzymes (DUBs) play important roles in many physiological and pathological processes by modulating the ubiquitination of their substrates. DUBs undergo post-translational modifications including ubiquitination. However, whether DUBs can reverse their own ubiquitination and regulate their own protein stability requires further investigation. To answer this question, we screened an expression library of DUBs and their enzymatic activity mutants and found that some DUBs regulated their own protein stability in an enzymatic activity- and homomeric interaction-dependent manner. Taking Ubiquitin-specific-processing protease 29 (USP29) as an example, we found that USP29 deubiquitinates itself and protects itself from proteasomal degradation. We also revealed that the N-terminal region of USP29 is critical for its protein stability. Taken together, our work demonstrates that at least some DUBs regulate their own ubiquitination and protein stability. Our findings provide novel molecular insight into the diverse regulation of DUBs.

The deubiquitinating enzyme OTUD1 antagonizes BH3-mimetic inhibitor induced cell death through regulating the stability of the MCL1 protein.

BACKGROUND: Myeloid cell leukaemia 1 (MCL1) is a pro-survival Bcl-2 family protein that plays important roles in cell survival, proliferation, differentiation and tumourigenesis. MCL1 is a fast-turnover protein that is degraded via an ubiquitination/proteasome-dependent mechanism. Although several E3 ligases have been discovered to promote the ubiquitination of MCL1, the deubiquitinating enzyme (DUB) that regulates its stability requires further investigation. METHODS: The immunoprecipitation was used to determine the interaction between OTUD1 and MCL1. The ubiquitination assays was performed to determine the regulation of MCL1 by OTUD1. The cell viability was used to determine the regulation of BH3-mimetic inhibitor induced cell death by OTUD1. The survival analysis was used to determine the relationship between OTUD1 expression levels and the survival rate of cancer patients. RESULTS: By screening a DUB expression library, we determined that the deubiquitinating enzyme OTUD1 regulates MCL1 protein stability in an enzymatic-activity dependent manner. OTUD1 interacts with MCL1 and promotes its deubiquitination. Knockdown of OTUD1 increases the sensitivity of tumour cells to the BH3-mimetic inhibitor ABT-263, while overexpression of OTUD1 increases tumour cell tolerance of ABT-263. Furthermore, bioinformatics analysis data reveal that OTUD1 is a negative prognostic factor for liver cancer, ovarian cancer and specific subtypes of breast and cervical cancer. CONCLUSIONS: The deubiquitinating enzyme OTUD1 antagonizes BH3-mimetic inhibitor induced cell death through regulating the stability of the MCL1 protein. Thus, OTUD1 could be considered as a therapeutic target for curing these cancers.

Ubiquitin-Specific Protease USP6 Regulates the Stability of the c-Jun Protein.

The c-Jun gene encodes a transcription factor that has been implicated in many physiological and pathological processes. c-Jun is a highly unstable protein that is degraded through a ubiquitination/proteasome-dependent mechanism. However, the deubiquitinating enzyme (DUB) that regulates the stability of the c-Jun protein requires further investigation. Here, by screening a DUB expression library, we identified ubiquitin-specific protease 6 (USP6) and showed that it regulates the stability of the c-Jun protein in a manner depending on its enzyme activity. USP6 interacts with c-Jun and antagonizes its ubiquitination. USP6 overexpression upregulates the activity of the downstream signaling pathway mediated by c-Jun/AP-1 and promotes cell invasion. Moreover, many aberrant genes that are upregulated in USP6 translocated nodular fasciitis are great potential targets regulated by c-Jun. Based on our data, USP6 is an enzyme that deubiquitinates c-Jun and regulates its downstream cellular functions.

Improving pairwise sequence alignment between distantly related proteins.

Sequence alignment between remotely related proteins has been one of the more difficult problems in structural biology. Improvements have been achieved by incorporating information that enhances the diversity of the substitution matrices. NdPASA is a web-based server that optimizes sequence alignments between proteins sharing low percentages of sequence identity. The program integrates structure information of the template sequence into a global alignment algorithm by employing amino acids' neighbor-dependent propensities for secondary structure as unique parameters for alignment. NdPASA optimizes alignment by evaluating the likelihood of a residue pair in the query sequence matching against a corresponding residue pair adopting a particular secondary structure in the template sequence. The server is designed to aid homologous protein structure modeling. It is most effective when the structure of the template sequence is known. NdPASA can be accessed online at www.fenglab.org/bioserver.html.

Computational exploration of the activated pathways associated with DNA damage response in breast cancer.

Molecular signaling events regulate cellular activity. Cancer stimulating signals trigger cellular responses that evade the regulatory control of cell development. To understand the mechanism of signaling regulation in cancer, it is necessary to identify the activated pathways in cancer. We have developed RepairPATH, a computational algorithm that explores the activated signaling pathways in cancer. The RepairPATH integrates RepairNET, an assembled protein interaction network associated with DNA damage response, with the gene expression profiles derived from the microarray data. Based on the observation that cofunctional proteins often exhibit correlated gene expression profiles, it identifies the activated signaling pathways in cancer by systematically searching the RepairNET for proteins with significantly correlated gene expression profiles. Analyzing the gene expression profiles of breast cancer, we found distinct similarities and differences in the activated signaling pathways between the samples from the patients who developed metastases and the samples from the patients who were disease free within 5 years. The cellular pathways associated with the various DNA repair mechanisms and the cell-cycle checkpoint controls are found to be activated in both sample groups. One of the most intriguing findings is that the pathways associated with different cellular processes are functionally coordinated through BRCA1 in the disease-free sample group, whereas such functional coordination is absent in the samples from patients who developed metastases. Our analysis revealed the potential cellular pathways that regulate the signaling events in breast cancer.

RepairNET: a bioinformatics toolbox for functional exploration of DNA damage response.

DNA damage response is one of the essential cellular mechanisms to maintaining the genomic integrity of the cell. Aberrations in the mechanism of DNA damage response often result in cancer. We describe here RepairNET, a protein-protein interaction network associated with the DNA damage response. RepairNET is assembled from the published literature by using a protocol that involved computational data mining of the MEDLINE and manual curation. This network represents the current knowledge on the intrinsic signaling pathways related to the DNA damage response process. RepairNET currently contains more than 1,200 proteins with over 2,300 functional interactions. A number of web-interface tools have been implemented to facilitate a user-friendly environment. The users can navigate through the cellular network associated with the DNA damage response via a Java-based interactive graphical interface. In order to help users explore the functional relationships between the interacting proteins, we have assigned functional domains to the proteins in RepairNET based on their sequences. A total of 365 unique functional domains are assigned. RepairNET is available online at http://guanyin.chem.temple.edu/RepairNET.html. It could become an essential resource center for cancer research, providing clues to understanding the functional relationship between proteins in the network, and to building scientific models for the mechanism of DNA damage response and cancer proliferation.

NdPASA: a pairwise sequence alignment server for distantly related proteins.

SUMMARY: NdPASA is a web server specifically designed to optimize sequence alignment between distantly related proteins. The program integrates structure information of the template sequence into a global alignment algorithm by employing neighbor-dependent propensities of amino acids as a unique parameter for alignment. NdPASA optimizes alignment by evaluating the likelihood of a residue pair in the query sequence matching against a corresponding residue pair adopting a particular secondary structure in the template sequence. NdPASA is most effective in aligning homologous proteins sharing low percentage of sequence identity. The server is designed to aid homologous protein structure modeling. A PSI-BLAST search engine was implemented to help users identify template candidates that are most appropriate for modeling the query sequences.

NdPASA: a novel pairwise protein sequence alignment algorithm that incorporates neighbor-dependent amino acid propensities.

Sequence alignment has become one of the essential bioinformatics tools in biomedical research. Existing sequence alignment methods can produce reliable alignments for homologous proteins sharing a high percentage of sequence identity. The performance of these methods deteriorates sharply for the sequence pairs sharing less than 25% sequence identity. We report here a new method, NdPASA, for pairwise sequence alignment. This method employs neighbor-dependent propensities of amino acids as a unique parameter for alignment. The values of neighbor-dependent propensity measure the preference of an amino acid pair adopting a particular secondary structure conformation. NdPASA optimizes alignment by evaluating the likelihood of a residue pair in the query sequence matching against a corresponding residue pair adopting a particular secondary structure in the template sequence. Using superpositions of homologous proteins derived from the PSI-BLAST analysis and the Structural Classification of Proteins (SCOP) classification of a nonredundant Protein Data Bank (PDB) database as a gold standard, we show that NdPASA has improved pairwise alignment. Statistical analyses of the performance of NdPASA indicate that the introduction of sequence patterns of secondary structure derived from neighbor-dependent sequence analysis clearly improves alignment performance for sequence pairs sharing less than 20% sequence identity. For sequence pairs sharing 13-21% sequence identity, NdPASA improves the accuracy of alignment over the conventional global alignment (GA) algorithm using the BLOSUM62 by an average of 8.6%. NdPASA is most effective for aligning query sequences with template sequences whose structure is known. NdPASA can be accessed online at http://astro.temple.edu/feng/Servers/BioinformaticServers.htm.

LINKER: a web server to generate peptide sequences with extended conformation.

LINKER was developed as an online server to assist biomedical researchers to design linker sequences for constructing functional fusion proteins. The program automatically generates a set of peptide sequences that are known to adopt extended conformations as determined by X-ray crystallography and NMR. In addition to the desired linker sequence length, the web interface provides a number of optional input parameters so that the users may enhance sequence selection based on the requirements of specific applications. The output of LINKER includes a list of peptide sequences with specified length and sequence characteristics. A graphical subroutine was implemented to highlight the chemical features of every linker sequence by hydrophobicity plots. LINKER can be accessed at http://astro.temple.edu/~feng/Servers/BioinformaticServers.htm.

Repair-FunMap: a functional database of proteins of the DNA repair systems.

UNLABELLED: Repair-FunMap is a functional database of the DNA repair systems. This database contains not only the proteins directly involved in DNA repair, but also the proteins that interact with the DNA repair proteins. A protein interaction network associated with the human DNA repair processes was established according to the functional relationship between proteins in the database. This network represents the current knowledge on the intrinsic signaling pathways related to DNA repair. The Repair-FunMap could become an essential resource center for cancer research, providing clues to understanding the inter-relationship between proteins in the network, and to building scientific models of the DNA repair processes. AVAILABILITY: http://astro.temple.edu/~feng/Servers/BioinformaticServers.htm

Exploring the sequence patterns in the alpha-helices of proteins.

This paper reports an extensive sequence analysis of the alpha-helices of proteins. alpha-Helices were extracted from the Protein Data Bank (PDB) and were divided into groups according to their sizes. It was found that some amino acids had differential propensity values for adopting helical conformation in short, medium and long alpha-helices. Pro and Trp had a significantly higher propensity for helical conformation in short helices than in medium and long helices. Trp was the strongest helix conformer in short helices. Sequence patterns favoring helical conformation were derived from a neighbor-dependent sequence analysis of proteins, which calculated the effect of neighboring amino acid type on the propensity of residues for adopting a particular secondary structure in proteins. This method produced an enhanced statistical significance scale that allowed us to explore the positional preference of amino acids for alpha-helical conformations. It was shown that the amino acid pair preference for alpha-helix had a unique pattern and this pattern was not always predictable by assuming proportional contributions from the individual propensity values of the amino acids. Our analysis also yielded a series of amino acid dyads that showed preference for alpha-helix conformation. The data presented in this study, along with our previous study on loop sequences of proteins, should prove useful for developing potential 'codes' for recognizing sequence patterns that are favorable for specific secondary structural elements in proteins.