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1.
Viruses ; 15(12)2023 12 13.
Article in English | MEDLINE | ID: mdl-38140657

ABSTRACT

St. Louis encephalitis virus (SLEV) is a neglected mosquito-borne Flavivirus that may cause severe neurological disease in humans and other animals. There are no specific treatments against SLEV infection or disease approved for human use, and drug repurposing may represent an opportunity to accelerate the development of treatments against SLEV. Here we present a scalable, medium-throughput phenotypic cell culture-based screening assay on Vero CCL81 cells to identify bioactive compounds that could be repurposed against SLEV infection. We screened eighty compounds from the Medicines for Malaria Venture (MMV) COVID Box library to identify nine (11%) compounds that protected cell cultures from SLEV-induced cytopathic effects, with low- to mid-micromolar potencies. We validated six hit compounds using viral plaque-forming assays to find that the compounds ABT-239, Amiodarone, Fluphenazine, Posaconazole, Triparanol, and Vidofludimus presented varied levels of antiviral activity and selectivity depending on the mammalian cell type used for testing. Importantly, we identified and validated the antiviral activity of the anti-flavivirus nucleoside analog 7DMA against SLEV. Triparanol and Fluphenazine reduced infectious viral loads in both Vero CCL81 and HBEC-5i cell cultures and, similar to the other validated compounds, are likely to exert antiviral activity through a molecular target in the host.


Subject(s)
Encephalitis, St. Louis , Flavivirus , Malaria , Triparanol , Animals , Humans , Encephalitis Virus, St. Louis , Encephalitis, St. Louis/diagnosis , Fluphenazine , Antiviral Agents/pharmacology , Mammals
2.
World J Biol Chem ; 12(3): 38-51, 2021 May 27.
Article in English | MEDLINE | ID: mdl-34084286

ABSTRACT

BACKGROUND: Tubulins, building blocks of microtubules, are modified substrates of diverse post-translational modifications including phosphorylation, polyglycylation and polyglutamylation. Polyglutamylation of microtubules, catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family, can regulate interactions with molecular motors and other proteins. Due to the diversity and functional importance of microtubule modifications, strict control of the TTLL enzymes has been suggested. AIM: To characterize the interaction between never in mitosis gene A-related kinase 5 (NEK5) and TTLL4 proteins and the effects of TTLL4 phosphorylation. METHODS: The interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5 (a.a. 260-708) as bait and confirmed by immunoprecipitation. The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced. RESULTS: Here, we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity. We further show that NEK5 can also interact with TTLL5 and TTLL7. The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4. The same effects were observed after the expression of the catalytically inactive form of NEK5. This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues. CONCLUSION: Our results demonstrate, for the first time, the regulation of TTLL activity through phosphorylation, pointing to NEK5 as a potential effector kinase. We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.

3.
Oncotarget ; 11(46): 4325-4337, 2020 Nov 17.
Article in English | MEDLINE | ID: mdl-33245729

ABSTRACT

Hyaluronic Acid-binding protein 4 (HABP4) is a regulatory protein of 57 kDa that is functionally involved in transcription regulation and RNA metabolism and shows several characteristics common to oncoproteins or tumor suppressors, including altered expression in cancer tissues, nucleus/cytoplasm shuttling, intrinsic lack of protein structure, complex interactomes and post translational modifications. Its gene has been found in a region on chromosome 9q22.3-31, which contains SNP haplotypes occurring in individuals with a high risk for familial colon cancer. To test a possible role of HABP4 in tumorigenesis we generated knockout mice by the CRISPR/Cas9 method and treated the animals with azoxymethane (AOM)/dextran sodium sulfate (DSS) for induction of colon tumors. HABP4-/- mice, compared to wild type mice, had more and larger tumors, and expressed more of the proliferation marker proteins Cyclin-D1, CDK4 and PCNA. Furthermore, the cells of the bottom of the colon crypts in the HABP4-/- mice divided more rapidly. Next, we generated also HABP4-/- HCT 116 cells, in cell culture and found again an increased proliferation in clonogenic assays in comparison to wild-type cells. Our study of the protein expression levels of HABP4 in human colon cancer samples, through immunohistochemistry assays, showed, that 30% of the tumors analyzed had low expression of HABP4. Our data suggest that HABP4 is involved in proliferation regulation of colon cells in vitro and in vivo and that it is a promising new candidate for a tumor suppressor protein that can be explored both in the diagnosis and possibly therapy of colon cancer.

4.
BMC Cancer ; 20(1): 23, 2020 Jan 06.
Article in English | MEDLINE | ID: mdl-31906878

ABSTRACT

BACKGROUND: The NEK serine/threonine protein kinases are involved in cell cycle checkpoints, DNA damage repair, and apoptosis. Alterations in these pathways are frequently associated with cell malignant cellular transformations. Thyroid cancer is the most common malignant tumour in the endocrine system. Despite good treatment methods, the number of cases has increased significantly in recent years. Here, we studied the expression of NEK1, NEK2, NEK3, and NEK5 in different types of normal and malignant tissues, using tissue microarray analysis, and identified NEKs as potential markers in thyroid malignancy. METHODS: The studied cases comprised multiple cancer tissue microarrays, including breast, colon, esophagus, kidney, lung, pancreas, prostate, stomach, thyroid and uterine cervix, as well as 281 patients who underwent thyroid resection for thyroid cancer or thyroid nodules. The expression of NEK1, NEK2, NEK3, and NEK5 was analyzed by immunohistochemistry. The expression pattern was evaluated in terms of intensity by two methods, semiquantitative and quantitative, and was compared between normal and cancer tissue. RESULTS: We analysed the expression of each member of the NEK family in a tissue-dependent manner. Compared to normal tissue, most of the evaluated proteins showed lower expression in lung tumour. However, in the thyroid, the expression was higher in malignant tissue, especially for NEK 1, NEK3 and NEK5. Concerning characteristics of the thyroid tumour, such as aggressiveness, NEK1 expression was higher in tumours with multifocality and in patients with lymph node metastasis. NEK3 expression was stronger in patients with stage II, that involved metastasis. NEK5, on the other hand, showed high expression in patients with invasion and metastasis and in patients with tumour size > 4 cm. Furthermore, this work, demonstrated for the first time a high specificity and sensitivity of over-expression of NEK1 in classical and follicular variants of papillary thyroid cancer and NEK3 in tall-cell papillary thyroid cancer. CONCLUSION: Taken together, the NEK protein kinases emerge as important proteins in thyroid cancer development and may help to identify malignancy and aggressiveness features during diagnosis. TRIAL REGISTRATION: This study was retrospectively registered.  www.accamargo.org.br/cientistas-pesquisadores/comite-de-etica-em-pequisa-cep.


Subject(s)
NIMA-Related Kinases/metabolism , Thyroid Gland/enzymology , Thyroid Neoplasms/enzymology , Adult , Biomarkers, Tumor/metabolism , Female , Humans , Immunohistochemistry , Male , Middle Aged , NIMA-Related Kinase 1/metabolism , Neoplasm Metastasis , Neoplasm Staging , Prognosis , Retrospective Studies , Thyroid Neoplasms/diagnosis , Thyroid Neoplasms/pathology
5.
World J Biol Chem ; 10(3): 44-64, 2019 Nov 21.
Article in English | MEDLINE | ID: mdl-31768228

ABSTRACT

The 57 kDa antigen recognized by the Ki-1 antibody, is also known as intracellular hyaluronic acid binding protein 4 and shares 40.7% identity and 67.4% similarity with serpin mRNA binding protein 1, which is also named CGI-55, or plasminogen activator inhibitor type-1-RNA binding protein-1, indicating that they might be paralog proteins, possibly with similar or redundant functions in human cells. Through the identification of their protein interactomes, both regulatory proteins have been functionally implicated in transcriptional regulation, mRNA metabolism, specifically RNA splicing, the regulation of mRNA stability, especially, in the context of the progesterone hormone response, and the DNA damage response. Both proteins also show a complex pattern of post-translational modifications, involving Ser/Thr phosphorylation, mainly through protein kinase C, arginine methylation and SUMOylation, suggesting that their functions and locations are highly regulated. Furthermore, they show a highly dynamic cellular localization pattern with localizations in both the cytoplasm and nucleus as well as punctuated localizations in both granular cytoplasmic protein bodies, upon stress, and nuclear splicing speckles. Several reports in the literature show altered expressions of both regulatory proteins in a series of cancers as well as mutations in their genes that may contribute to tumorigenesis. This review highlights important aspects of the structure, interactome, post-translational modifications, sub-cellular localization and function of both regulatory proteins and further discusses their possible functions and their potential as tumor markers in different cancer settings.

6.
J Cell Biochem ; 120(10): 16853-16866, 2019 10.
Article in English | MEDLINE | ID: mdl-31090963

ABSTRACT

Cells are daily submitted to high levels of DNA lesions that trigger complex pathways and cellular responses by cell cycle arrest, apoptosis, alterations in transcriptional response, and the onset of DNA repair. Members of the NIMA-related kinase (NEK) family have been related to DNA damage response and repair and the first insight about NEK5 in this context is related to its role in centrosome separation resulting in defects in chromosome integrity. Here we investigate the potential correlation between NEK5 and the DNA damage repair index. The effect of NEK5 in double-strand breaks caused by etoposide was accessed by alkaline comet assay and revealed that NEK5-silenced cells are more sensitive to etoposide treatment. Topoisomerase IIß (TOPIIß) is a target of etoposide that leads to the production of DNA breaks. We demonstrate that NEK5 interacts with TOPIIß, and the dynamics of this interaction is evaluated by proximity ligation assay. The complex NEK5/TOPIIß is formed immediately after etoposide treatment. Taken together, the results of our study reveal that NEK5 depletion increases DNA damage and impairs proper DNA damage response, pointing out NEK5 as a potential kinase contributor to genomic stability.


Subject(s)
DNA Damage/genetics , DNA Repair/genetics , DNA Topoisomerases, Type II/metabolism , Etoposide/pharmacology , NIMA-Related Kinases/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Topoisomerase II Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Survival/drug effects , DNA/drug effects , DNA/genetics , HEK293 Cells , Humans , NIMA-Related Kinases/genetics , RNA Interference , RNA, Small Interfering/genetics
7.
World J Biol Chem ; 5(2): 141-60, 2014 May 26.
Article in English | MEDLINE | ID: mdl-24921005

ABSTRACT

Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A (NIMA)-related kinases (Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups' functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology.

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