A new concept to target the proteasome: disrupting protein-protein interactions in the proteasome super-assembly as a way to stop the growth of cancer cells.

ABSTRACTDe novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: thymidylate synthase (TYMS), serine hydroxymethyltransferase (SHMT) and dihydrofolate reductase (DHFR), targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, provide a useful tool to develop drugs targeting the entire complex instead of the individual components.

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BIRC3 and BIRC5: multi‐faceted inhibitors in cancer

Cell & Bioscience ◽

10.1186/s13578-020-00521-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Raffaele Frazzi

Keyword(s):

Cancer Cells ◽

Protein Interactions ◽

Point Mutations ◽

Therapeutic Targets ◽

Progression Free Survival ◽

Lymphocytic Leukemia ◽

Low Grade ◽

Main Body ◽

Protein Protein Interactions ◽

Smac Mimetics

Abstract Background The evasion from apoptosis is a common strategy adopted by most tumors, and inhibitors of apoptosis proteins (IAPs) are among the most studied molecular and therapeutic targets. BIRC3 (cellular IAP2) and BIRC5 (survivin) are two of the eight members of the human IAPs family. This family is characterized by the presence of the baculoviral IAP repeat (BIR) domains, involved in protein-protein interactions. In addition to the BIR domains, IAPs also contain other important domains like the C-terminal ubiquitin-conjugating (UBC) domain, the caspase recruitment (CARD) domain and the C-terminal Ring zinc-finger (RING) domain. Main body BIRC3 and BIRC5 have been characterized in some solid and hematological tumors and are therapeutic targets for the family of drugs called “Smac mimetics”. Many evidences point to the pro-survival and antiapoptotic role of BIRC3 in cancer cells, however, not all the data are consistent and the resulting picture is heterogeneous. For instance, BIRC3 genetic inactivation due to deletions or point mutations is consistently associated to shorter progression free survival and poor prognosis in chronic lymphocytic leukemia patients. BIRC3 inactivation has also been associated to chemoimmunotherapy resistance. On the contrary, the progression from low grade gliomas to high grade gliomas is accompanied by BIRC3 expression increase, which bears relevant prognostic consequences. Due to the relationship between BIRC3, MAP3K14 and the non-canonical NF-kB pathway, BIRC3 inactivation bears consequences also on the tumor cells relying on NF-kB pathway to survive. BIRC5, on the contrary, is commonly considered an anti-apoptotic molecule, promoting cell division and tumor progression and it is widely regarded as potential therapeutic target. Conclusions The present manuscript collects and reviews the most recent literature concerning the role played by BIRC3 and BIRC5 in cancer cells, providing useful information for the choice of the best therapeutic targets.

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Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007213117 ◽

2020 ◽

Vol 117 (47) ◽

pp. 29684-29690

Author(s):

Matthias Barone ◽

Matthias Müller ◽

Slim Chiha ◽

Jiang Ren ◽

Dominik Albat ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Protein Interactions ◽

Breast Cancer Cells ◽

Cancer Metastasis ◽

Cancer Therapies ◽

Protein Protein Interactions ◽

Zebrafish Model ◽

Therapeutic Concept ◽

Protein Protein Interaction

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor (Kd=120 nM,MW=734Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

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How Cancer Cells Resist Chemotherapy: Design and Development of Drugs Targeting Protein-Protein Interactions

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190305130141 ◽

2019 ◽

Vol 19 (6) ◽

pp. 394-412 ◽

Cited By ~ 5

Author(s):

Vadim V. Tarasov ◽

Vladimir N. Chubarev ◽

Ghulam Md Ashraf ◽

Samira A. Dostdar ◽

Alexander V. Sokolov ◽

...

Keyword(s):

Drug Resistance ◽

Clinical Outcome ◽

Cancer Cells ◽

Protein Interactions ◽

Resistance Mechanisms ◽

Protein Protein Interactions ◽

Treatment Regimens ◽

Pubmed Search ◽

Bona Fide ◽

Improve Clinical Outcome

Background:Resistance toward chemotherapeutics is one of the main obstacles on the way to effective cancer treatment. Personalization of chemotherapy could improve clinical outcome. However, despite preclinical significance, most of the potential markers have failed to reach clinical practice partially due to the inability of numerous studies to estimate the marker’s impact on resistance properly.Objective:The analysis of drug resistance mechanisms to chemotherapy in cancer cells, and the proposal of study design to identify bona fide markers.Methods:A review of relevant papers in the field. A PubMed search with relevant keywords was used to gather the data. An example of a search request: drug resistance AND cancer AND paclitaxel.Results:We have described a number of drug resistance mechanisms to various chemotherapeutics, as well as markers to underlie the phenomenon. We also proposed a model of a rational-designed study, which could be useful in determining the most promising potential biomarkers.Conclusion:Taking into account the most reasonable biomarkers should dramatically improve clinical outcome by choosing the suitable treatment regimens. However, determining the leading biomarkers, as well as validating of the model, is a work for further investigations.

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Analysis of Protein Biology to Increase Efficacy of Aurora Kinase Inhibitors for Multiple Myeloma Therapy.

Blood ◽

10.1182/blood.v112.11.1699.1699 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1699-1699

Author(s):

Robert P. Evans ◽

Kim Laginha ◽

Linda M. Pilarski ◽

Andrew Belch ◽

Tony Reiman

Keyword(s):

Multiple Myeloma ◽

Cancer Cells ◽

Protein Interactions ◽

Kinase Inhibitors ◽

Aurora Kinase ◽

Protein Protein Interactions ◽

Aurora Kinases ◽

Aurora Kinase Inhibitors ◽

Anti Cancer ◽

Novel Protein

Abstract Despite improvement in therapeutic regimes which have extended survival of patients with multiple myeloma, the search for novel targets to further advance treatment options continues. It is critical to the success of targeted cancer therapy that the rational selection of drugs be derived from the molecular and cellular biology of protein targets. Furthermore, studies have indicated that focused combination drug therapy could produce remarkable improvement in patient outcomes. Aurora kinases A/B/C (AURK), components of the centrosome and important for cell cycle function, are promising new targets in the treatment of numerous types of cancer. We have previously validated AURKs as a therapeutic target in pre-clinical models of multiple myeloma. In this study our aim is to better define the biology of AURKs in both normal and malignant cells, with the goal of identifying rational strategies for improving the therapeutic potential of Aurora kinase inhibitors (AKIs) as anti-cancer agents. We use a systematic two-pronged approach to map post-translational modifications and novel protein-protein interactions that define the interactome of AURKs. We also examine whether Aurora kinase interactions and posttranslational modifications are significantly different in cancer cells compared to normal cells. Increased understanding of Aurora kinase biology will help develop mechanisms to intensify the anti-cancer effects of AKIs. SUMOylation is a recently discovered posttranslational modification of numerous proteins that has been linked to many cancer types. A small ubiquitin-like modifier (SUMO) group is conjugated at the canonical ubiquitin site of a protein to alter its function, stability or sub-cellular localization. We believe that Aurora kinases may be differentially SUMOylated in cancer cells compared to normal cells, and that SUMOylation of Aurora kinases in cancer cells may alter their function and their susceptibility to AKI therapy. Here we have determined putative SUMOylation sites of all three AURKs in silico and also have evidence of AURK SUMOylation using an in vitro assay and also in human HEK293 cells. Furthermore, the gene encoding the central SUMO conjugating enzyme - UBC9 - is expressed at higher levels in CD138+ malignant plasma cells compared to other CD138−bone marrow cells in myeloma patients. As part of our strategy, we have also used Tandem Affinity Purification (TAP) technology to identify novel protein-protein interactions of Aurora kinases. In the initial stage, we have established stable expressing clones in a human cancer cell line and a normal line that express TAP-tagged AURKA/B or C. In pilot experiments we have successfully isolated pure recombinant AURKA along with interacting proteins using the TAP method. Using mass spectrometry (MS/MS) protein ID, we have identified putative protein interaction partners with AURKA which have implications in general cancer biology and centrosomal structure. In the future, these interactions will be validated using conventional biochemical methods before being tested in myeloma cell lines.

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Profiling protein–protein interactions of single cancer cells with in situ lysis and co-immunoprecipitation

Lab on a Chip ◽

10.1039/c9lc00139e ◽

2019 ◽

Vol 19 (11) ◽

pp. 1922-1928 ◽

Cited By ~ 1

Author(s):

Ji Young Ryu ◽

Jihye Kim ◽

Min Ju Shon ◽

Jiashu Sun ◽

Xingyu Jiang ◽

...

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Target Proteins ◽

Single Cancer

We developed a single-cell version of the co-immunoprecipitation (co-IP) analysis that examines the amount and protein–protein interactions of target proteins immunoprecipitated from individual cells.

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Identification of Cross-Pathway Connections via Protein-Protein Interactions Linked to Altered States of Metabolic Enzymes in Cervical Cancer

Frontiers in Medicine ◽

10.3389/fmed.2021.736495 ◽

2021 ◽

Vol 8 ◽

Author(s):

Krishna Kumar ◽

Sarpita Bose ◽

Saikat Chakrabarti

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Signaling Pathway ◽

Protein Interactions ◽

Interaction Network ◽

Metabolic Enzymes ◽

Metabolic Reprogramming ◽

Signaling Proteins ◽

Altered States ◽

Protein Protein Interactions

Metabolic reprogramming is one of the emerging hallmarks of cancer cells. Various factors, such as signaling proteins (S), miRNA, and transcription factors (TFs), may play important roles in altering the metabolic status in cancer cells by interacting with metabolic enzymes either directly or via protein-protein interactions (PPIs). Therefore, it is important to understand the coordination among these cellular pathways, which may provide better insight into the molecular mechanism behind metabolic adaptations in cancer cells. In this study, we have designed a cervical cancer-specific supra-interaction network where signaling pathway proteins, TFs, and microRNAs (miRs) are connected to metabolic enzymes via PPIs to investigate novel molecular targets and connections/links/paths regulating the metabolic enzymes. Using publicly available omics data and PPIs, we have developed a Hidden Markov Model (HMM)-based mathematical model yielding 94, 236, and 27 probable links/paths connecting signaling pathway proteins, TFs, and miRNAs to metabolic enzymes, respectively, out of which 83 paths connect to six common metabolic enzymes (RRM2, NDUFA11, ENO2, EZH2, AKR1C2, and TYMS). Signaling proteins (e.g., PPARD, BAD, GNB5, CHECK1, PAK2, PLK1, BRCA1, MAML3, and SPP1), TFs (e.g., KAT2B, ING1, MED1, ZEB1, AR, NCOA2, EGR1, TWIST1, E2F1, ID4, RBL1, ESR1, and HSF2), and miR (e.g., mir-147a, mir-593-5p, mir-138-5p, mir-16-5p, and mir-15b-5p) were found to regulate two key metabolic enzymes, EZH2 and AKR1C2, with altered metabolites (L-lysine and tetrahydrodeoxycorticosterone, THDOC) status in cervical cancer. We believe, the biology-based approach of our system will pave the way for future studies, which could be aimed toward identifying novel signaling, transcriptional, and post-transcriptional regulators of metabolic alterations in cervical cancer.

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