Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

The Mitochondrial Connection: The Nek Kinases' New Functional Axis in Mitochondrial Homeostasis.

Mitochondria provide energy for all cellular processes, including reactions associated with cell cycle progression, DNA damage repair, and cilia formation. Moreover, mitochondria participate in cell fate decisions between death and survival. Nek family members have already been implicated in DNA damage response, cilia formation, cell death, and cell cycle control. Here, we discuss the role of several Nek family members, namely Nek1, Nek4, Nek5, Nek6, and Nek10, which are not exclusively dedicated to cell cycle-related functions, in controlling mitochondrial functions. Specifically, we review the function of these Neks in mitochondrial respiration and dynamics, mtDNA maintenance, stress response, and cell death. Finally, we discuss the interplay of other cell cycle kinases in mitochondrial function and vice versa. Nek1, Nek5, and Nek6 are connected to the stress response, including ROS control, mtDNA repair, autophagy, and apoptosis. Nek4, in turn, seems to be related to mitochondrial dynamics, while Nek10 is involved with mitochondrial metabolism. Here, we propose that the participation of Neks in mitochondrial roles is a new functional axis for the Nek family.

NEK6 Regulates Redox Balance and DNA Damage Response in DU-145 Prostate Cancer Cells.

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy.

Identification of biological pathways and processes regulated by NEK5 in breast epithelial cells via an integrated proteomic approach.

Specific members of the Nima-Related Kinase (NEK) family have been linked to cancer development and progression, and a role for NEK5, one of the least studied members, in breast cancer has recently been proposed. However, while NEK5 is known to regulate centrosome separation and mitotic spindle assembly, NEK5 signalling mechanisms and function in this malignancy require further characterization. To this end, we established a model system featuring overexpression of NEK5 in the immortalized breast epithelial cell line MCF-10A. MCF-10A cells overexpressing NEK5 exhibited an increase in clonogenicity under monolayer conditions and enhanced acinar size and abnormal morphology in 3D Matrigel culture. Interestingly, they also exhibited a marked reduction in Src activation and downstream signalling. To interrogate NEK5 signalling and function in an unbiased manner, we applied a variety of MS-based proteomic approaches. Determination of the NEK5 interactome by Bio-ID identified a variety of protein classes including the kinesins KIF2C and KIF22, the mitochondrial proteins TFAM, TFB2M and MFN2, RhoH effectors and the negative regulator of Src, CSK. Characterization of proteins and phosphosites modulated upon NEK5 overexpression by global MS-based (phospho)proteomic profiling revealed impact on the cell cycle, DNA synthesis and repair, Rho GTPase signalling, the microtubule cytoskeleton and hemidesmosome assembly. Overall, the study indicates that NEK5 impacts diverse pathways and processes in breast epithelial cells, and likely plays a multifaceted role in breast cancer development and progression. Video Abstract.

The Isoflavanoid (+)-PTC Regulates Cell-Cycle Progression and Mitotic Spindle Assembly in a Prostate Cancer Cell Line.

Prostate cancer is the second most common malignancy in men and the development of effective therapeutic strategies remains challenging when more advanced, androgen-independent or insensitive forms are involved. Accordingly, we have evaluated, using flow cytometry, confocal microscopy and image analysis, the anti-proliferative effects of (+)-2,3,9-trimethoxypterocarpan [(+)-PTC, 1] on relevant human prostate cancer cells as well as its capacity to control mitosis within them. In particular, the studies reported herein reveal that (+)-PTC exerts anti-proliferative activity against the PC-3 cell lines by regulating cell-cycle progression with mitosis being arrested in the prophase or prometaphase. Furthermore, it emerges that treatment of the target cells with this compound results in the formation of monopolar spindles, disorganized centrosomes and extensively disrupted γ-tubulin distributions while centriole replication remains unaffected. Such effects suggest (+)-PTC should be considered as a possible therapy for androgen-insensitive/independent prostate cancer.

Nek4 regulates mitochondrial respiration and morphology.

Nek4 is a serine/threonine kinase which has been implicated in primary cilia stabilization, DNA damage response, autophagy and epithelial-to-mesenchymal transition. The role of Nek4 in cancer cell survival and chemotherapy resistance has also been shown. However, the precise mechanisms by which Nek4 operates remain to be elucidated. Here, we show that Nek4 overexpression activates mitochondrial respiration coupled to ATP production, which is paralleled by increased mitochondrial membrane potential, and resistance to mitochondrial DNA damage. Congruently, Nek4 depletion reduced mitochondrial respiration and mtDNA integrity. Nek4 deficiency caused mitochondrial elongation, probably via reduced activity of the fission protein DRP1. In Nek4 overexpressing cells, the increase in mitochondrial fission was concomitant to enhanced phosphorylation of DRP1 and Erk1/2 proteins, and the effects on mitochondrial respiration were abolished in the presence of a DRP1 inhibitor. This study shows Nek4 as a novel regulator of mitochondrial function that may explain the joint appearance of high mitochondrial respiration and mitochondrial fragmentation.

Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A -related kinase 5.

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.

Using the E1A Minigene Tool to Study mRNA Splicing Changes.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5´capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response.

NIMA-related kinases, or NEKs, are a family of Ser/Thr protein kinases involved in cell cycle and mitosis, centrosome disjunction, primary cilia functions, and DNA damage responses among other biological functional contexts in vertebrate cells. In human cells, there are 11 members, termed NEK1 to 11, and the research has mainly focused on exploring the more predominant roles of NEKs in mitosis regulation and cell cycle. A possible important role of NEKs in DNA damage response (DDR) first emerged for NEK1, but recent studies for most NEKs showed participation in DDR. A detailed analysis of the protein interactions, phosphorylation events, and studies of functional aspects of NEKs from the literature led us to propose a more general role of NEKs in DDR. In this review, we express that NEK1 is an activator of ataxia telangiectasia and Rad3-related (ATR), and its activation results in cell cycle arrest, guaranteeing DNA repair while activating specific repair pathways such as homology repair (HR) and DNA double-strand break (DSB) repair. For NEK2, 6, 8, 9, and 11, we found a role downstream of ATR and ataxia telangiectasia mutated (ATM) that results in cell cycle arrest, but details of possible activated repair pathways are still being investigated. NEK4 shows a connection to the regulation of the nonhomologous end-joining (NHEJ) repair of DNA DSBs, through recruitment of DNA-PK to DNA damage foci. NEK5 interacts with topoisomerase IIß, and its knockdown results in the accumulation of damaged DNA. NEK7 has a regulatory role in the detection of oxidative damage to telomeric DNA. Finally, NEK10 has recently been shown to phosphorylate p53 at Y327, promoting cell cycle arrest after exposure to DNA damaging agents. In summary, this review highlights important discoveries of the ever-growing involvement of NEK kinases in the DDR pathways. A better understanding of these roles may open new diagnostic possibilities or pharmaceutical interventions regarding the chemo-sensitizing inhibition of NEKs in various forms of cancer and other diseases.

NEK1 deficiency affects mitochondrial functions and the transcriptome of key DNA repair pathways.

Previous studies have indicated important roles for NIMA-related kinase 1 (NEK1) in modulating DNA damage checkpoints and DNA repair capacity. To broadly assess the contributions of NEK1 to genotoxic stress and mitochondrial functions, we characterised several relevant phenotypes of NEK1 CRISPR knockout (KO) and wild-type (WT) HAP1 cells. Our studies revealed that NEK1 KO cells resulted in increased apoptosis and hypersensitivity to the alkylator methyl methanesulfonate, the radiomimetic bleomycin and UVC light, yet increased resistance to the crosslinker cisplatin. Mitochondrial functionalities were also altered in NEK1 KO cells, with phenotypes of reduced mitophagy, increased total mitochondria, elevated levels of reactive oxygen species, impaired complex I activity and higher amounts of mitochondrial DNA damage. RNA-seq transcriptome analysis coupled with quantitative real-time PCR studies comparing NEK1 KO cells with NEK1 overexpressing cells revealed that the expression of genes involved in DNA repair pathways, such as base excision repair, nucleotide excision repair and double-strand break repair, are altered in a way that might influence genotoxin resistance. Together, our studies underline and further support that NEK1 serves as a hub signalling kinase in response to DNA damage, modulating DNA repair capacity, mitochondrial activity and cell fate determination.

NEK5 interacts with LonP1 and its kinase activity is essential for the regulation of mitochondrial functions and mtDNA maintenance.

Little is known about Nima-related kinase (NEKs), a widely conserved family of kinases that have key roles in cell-cycle progression. Nevertheless, it is now clear that multiple NEK family members act in networks, not only to regulate specific events of mitosis, but also to regulate metabolic events independently of the cell cycle. NEK5 was shown to act in centrosome disjunction, caspase-3 regulation, myogenesis, and mitochondrial respiration. Here, we demonstrate that NEK5 interacts with LonP1, an AAA+ mitochondrial protease implicated in protein quality control and mtDNA remodeling, within the mitochondria and it might be involved in the LonP1-TFAM signaling module. Moreover, we demonstrate that NEK5 kinase activity is required for maintaining mitochondrial mass and functionality and mtDNA integrity after oxidative damage. Taken together, these results show a new role of NEK5 in the regulation of mitochondrial homeostasis and mtDNA maintenance, possibly due to its interaction with key mitochondrial proteins, such as LonP1.

Intracellular hyaluronic acid-binding protein 4 (HABP4): a candidate tumor suppressor in colorectal cancer.

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.

NEK10 interactome and depletion reveal new roles in mitochondria.

BACKGROUND: Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. METHODS: HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. RESULTS: Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. CONCLUSIONS: Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

Checking NEKs: Overcoming a Bottleneck in Human Diseases.

In previous years, several kinases, such as phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), and extracellular-signal-regulated kinase (ERK), have been linked to important human diseases, although some kinase families remain neglected in terms of research, hiding their relevance to therapeutic approaches. Here, a review regarding the NEK family is presented, shedding light on important information related to NEKs and human diseases. NEKs are a large group of homologous kinases with related functions and structures that participate in several cellular processes such as the cell cycle, cell division, cilia formation, and the DNA damage response. The review of the literature points to the pivotal participation of NEKs in important human diseases, like different types of cancer, diabetes, ciliopathies and central nervous system related and inflammatory-related diseases. The different known regulatory molecular mechanisms specific to each NEK are also presented, relating to their involvement in different diseases. In addition, important information about NEKs remains to be elucidated and is highlighted in this review, showing the need for other studies and research regarding this kinase family. Therefore, the NEK family represents an important group of kinases with potential applications in the therapy of human diseases.

Expression of the NEK family in normal and cancer tissue: an immunohistochemical study.

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.

Complex interactomes and post-translational modifications of the regulatory proteins HABP4 and SERBP1 suggest pleiotropic cellular functions.

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.

Insights into the full-length SRPK2 structure and its hydrodynamic behavior.

The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (~50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated ΔNΔS-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to-closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain.

NEK5 interacts with topoisomerase IIß and is involved in the DNA damage response induced by etoposide.

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.

Fasciculation and elongation zeta proteins 1 and 2: From structural flexibility to functional diversity.

Fasciculation and elongation zeta/zygin (FEZ) proteins are a family of hub proteins and share many characteristics like high connectivity in interaction networks, they are involved in several cellular processes, evolve slowly and in general have intrinsically disordered regions. In 1985, unc-76 gene was firstly described and involved in axonal growth in C. elegans, and in 1997 Bloom and Horvitz enrolled also the human homologues genes, FEZ1 and FEZ2, in this process. While nematodes possess one gene (unc-76), mammalians have one more copy (FEZ1 and FEZ2). Several animal models have been used to study FEZ family functions like: C. elegans, D. melanogaster, R. novergicus and human cells. Complementation assays were performed and demonstrated the function conservation between paralogues. Human FEZ1 protein is more studied followed by UNC-76 and FEZ2 proteins, respectively. While FEZ1 and UNC-76 shared interaction partners, FEZ2 evolved and increased the number of protein-protein interactions (PPI) with cytoplasmatic partners. FEZ proteins are implicated in intracellular transport, acting as bivalent cargo transport adaptors in kinesin-mediated movement. Especially in light of this cellular function, this family of proteins has been involved in several processes like neuronal development, neurological disorders, viral infection and autophagy. However, nuclear functions of FEZ proteins have been explored as well, due to high content of PPI with nuclear proteins, correlating FEZ1 expression to Sox2 and Hoxb4 gene regulation and retinoic acid signaling. These recent findings open new avenue to study FEZ proteins functions and its involvement in already described processes. This review intends to reunite aspects of evolution, structure, interaction partners and function of FEZ proteins and correlate them to physiological and pathological processes.

Fasciculation and elongation zeta-1 protein (FEZ1) interacts with the retinoic acid receptor and participates in transcriptional regulation of the Hoxb4 gene.

Fasciculation and elongation zeta-1 (FEZ1) protein is involved in axon outgrowth and is highly expressed in the brain. It has multiple interaction partners, with functions varying from the regulation of neuronal development and intracellular transport mechanisms to transcription regulation. One of its interactors is retinoic acid receptor (RAR), which is activated by retinoic acid and controls many target genes and physiological process. Based on previous evidence suggesting a possible nuclear role for FEZ1, we wanted to deepen our understanding of this function by addressing the FEZ1-RAR interaction. We performed in vitro binding experiments and assessed the interface of interaction between both proteins. We found that FEZ1-RAR interacted with a similar magnitude as RAR to its responsive element DR5 and that the interaction occurred in the coiled-coil region of FEZ1 and in the ligand-binding domain of RAR. Furthermore, cellular experiments were performed in order to confirm the interaction and screen for induced target genes from an 86-gene panel. The analysis of gene expression showed that only in the presence of retinoic acid did FEZ1 induce hoxb4 gene expression. This finding is consistent with data from the literature showing the hoxb4 gene functionally involved in development and acute myeloid leukemia, as is FEZ1.