bims-micesi 2022-03-27 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–03–27
nineteen papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Spring-like behavior of cytoplasm holds the mitotic spindle in place.
In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada).
MDC1 is essential for G2/M transition and spindle assembly in mouse oocytes.
Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase.
Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.
XBP1 variant 1 (Xv1) promotes mitosis of cancer cells involving upregulation of the polyglutamylase TTLL6.
Microtubule Anchoring: Attaching Dynamic Polymers to Cellular Structures.
Multiple actions of NMS-P715, the monopolar spindle 1 (MPS1) mitotic checkpoint inhibitor in liver fluke-associated cholangiocarcinoma cells.
Role of NIMA-related Kinase 2 (NEK2) in lung cancer: Mechanisms and therapeutic prospects.
Profilin is involved in G1 to S phase progression and mitotic spindle orientation during Leishmania donovani cell division cycle.
Phosphorylation of MAD2 at Ser195 Promotes Spindle Checkpoint Defects and Sensitizes Cancer Cells to Radiotherapy in ATM Deficient Cells.
CENP-B-mediated DNA loops regulate activity and stability of human centromeres.
Current progress and perspectives of CDC20 in female reproductive cancers.
SUMO-mediated recruitment allows timely function of the Yen1 nuclease in mitotic cells.
The kinesin Eg5 inhibitor K858 exerts antiproliferative and proapoptotic effects and attenuates the invasive potential of head and neck squamous carcinoma cells.
Pharmacophore optimization of imidazole chalcones to modulate microtubule dynamics.
Midkine inhibitor (iMDK) induces apoptosis of primary effusion lymphoma via G2/M cell cycle arrest.
Selective Recognition of Phosphatidylinositol Phosphate Receptors by C-Terminal Tail of Mitotic Kinesin-like Protein 2 (MKlp2).
Leveraging substrate stiffness to promote stem cell asymmetric division via mechanotransduction-polarity protein axis and its Bayesian regression analysis.

Proc Natl Acad Sci U S A. 2022 Apr 05. 119(14): e2203036119

Spring-like behavior of cytoplasm holds the mitotic spindle in place.

Luolan Bai, Timothy J Mitchison.

DOI: https://doi.org/10.1073/pnas.2203036119
Eur J Cell Biol. 2022 Mar 17. pii: S0171-9335(22)00020-6. [Epub ahead of print]101(2): 151217

In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada).

Pavla Tůmová, Eva Nohýnková, Gerhard Wanner.

  To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.

Keywords:  FIB/SEM; Giardia; Kinetochore; Mitosis; Nuclear envelope; Spindle apparatus

DOI:  https://doi.org/10.1016/j.ejcb.2022.151217
Cell Mol Life Sci. 2022 Mar 23. 79(4): 200

MDC1 is essential for G2/M transition and spindle assembly in mouse oocytes.

Jiyeon Leem, Jeong Su Oh.

  Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the ATM-Chk1/2-Cdc25 pathway, oocytes readily enter M-phase immediately following DNA damage. This implies a lack of a robust canonical G2/M DNA damage checkpoint in oocytes. Here we show that MDC1 plays a non-canonical role in controlling G2/M transition by regulating APC/C-Cdh1-mediated cyclin B1 degradation in response to DNA damage in mouse oocytes. Depletion of MDC1 impaired M-phase entry by decreasing cyclin B1 levels via the APC/C-Cdh1 pathway. Notably, the APC/C-Cdh1 regulation mediated by MDC1 was achieved by a direct interaction between MDC1 and APC/C-Cdh1. This interaction was transiently disrupted after DNA damage with a concomitant increase in Cdh1 levels, which, in turn, decreased cyclin B1 levels and delayed M-phase entry. Moreover, MDC1 depletion impaired spindle assembly by decreasing the integrity of microtubule organizing centers (MTOCs). Therefore, our results demonstrate that MDC1 is an essential molecule in regulating G2/M transition in response to DNA damage and in regulating spindle assembly in mouse oocytes. These results provide new insights into the regulation of the G2/M DNA damage checkpoint and cell cycle control in oocytes.

Keywords:  APC/C-Cdh1; DNA damage; G2/M transition; MDC1; Oocytes; Spindle assembly

DOI:  https://doi.org/10.1007/s00018-022-04241-1
Cell Rep. 2022 Mar 22. pii: S2211-1247(22)00298-4. [Epub ahead of print]38(12): 110554

Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase.

Drisya Vijayakumari, Janina Müller, Silke Hauf.

  Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and can cooperate with the proteasome for their degradation. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase, resulting in co-degradation of separase along with securin. If true, Cdc48 would be important in mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

Keywords:  CP: Cell Biology; Cdc48; TORC1; Ufd1; Ufd2; chromosome segregation; mitosis; securin; separase; ubiquitination

DOI:  https://doi.org/10.1016/j.celrep.2022.110554
Development. 2022 Mar 21. pii: dev.198341. [Epub ahead of print]

Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Kelsey E Brooks, Brittany L Daughtry, Brett Davis, Melissa Y Yan, Suzanne S Fei, Selma Shepherd, Lucia Carbone, Shawn L Chavez.

  Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure, or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus, or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions, and asymmetric genome partitioning was also revealed and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein, BUB1B/BUBR1, resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network mediating mitotic progression, all prior to zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally-derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

Keywords:  Aneuploidy; BUB1B/BUBR1; Cytokinesis; Embryo; Micronuclei; Mitosis

DOI:  https://doi.org/10.1242/dev.198341
Hum Mol Genet. 2022 Mar 25. pii: ddac010. [Epub ahead of print]

XBP1 variant 1 (Xv1) promotes mitosis of cancer cells involving upregulation of the polyglutamylase TTLL6.

Yongwang Zhong, Wenjing Yan, Jingjing Ruan, Mike Fang, Changjun Yu, Shaojun Du, Ganesha Rai, Dingyin Tao, Mark J Henderson, Shengyun Fang.

XBP1 variant 1 (Xv1) is the most abundant XBP1 variant and is highly enriched across cancer types but nearly none in normal tissues. Its expression is associated with poor patients survival and is specifically required for survival of malignant cells, but the underlying mechanism is not known. Here we report that Xv1 upregulates the polyglutamylase Tubulin Tyrosine Ligase-Like 6 (TTLL6) and promotes mitosis of cancer cells. Like the canonical XBP1, Xv1 mRNA undergoes unconventional splicing by IRE1α under ER stress, but it is also constitutively spliced by IRE1β. The spliced Xv1 mRNA encodes the active form of Xv1 protein (Xv1s). RNA-seq in HeLa cells revealed that Xv1s overexpression regulates expression of genes that are not involved in the canonical UPR, including TTLL6 as a highly upregulated gene. Gel shift assay and chromatin immunoprecipitation revealed that Xv1s binds to the TTLL6 promoter region. Knockdown of TTLL6 caused death of cancer cells but not benign and normal cells, similar to the effects of knocking down Xv1. Moreover, overexpression of TTLL6 partially rescued BT474 cells from apoptosis induced by either TTLL6 or Xv1 knockdown, supporting TTLL6 as an essential downstream effector of Xv1 in regulating cancer cell survival. TTLL6 is localized in the mitotic spindle of cancer cells. Xv1 or TTLL6 knockdown resulted in decreased spindle polyglutamylation and interpolar spindle, as well as congression failure, mitotic arrest, and cell death. These findings suggest that Xv1 is essential for cancer cell mitosis, which is mediated, at least in part, by increasing TTLL6 expression.

DOI: https://doi.org/10.1093/hmg/ddac010
Front Cell Dev Biol. 2022 ;10 867870

Microtubule Anchoring: Attaching Dynamic Polymers to Cellular Structures.

Chithran Vineethakumari, Jens Lüders.

  Microtubules are dynamic, filamentous polymers composed of α- and β-tubulin. Arrays of microtubules that have a specific polarity and distribution mediate essential processes such as intracellular transport and mitotic chromosome segregation. Microtubule arrays are generated with the help of microtubule organizing centers (MTOC). MTOCs typically combine two principal activities, the de novo formation of microtubules, termed nucleation, and the immobilization of one of the two ends of microtubules, termed anchoring. Nucleation is mediated by the γ-tubulin ring complex (γTuRC), which, in cooperation with its recruitment and activation factors, provides a template for α- and β-tubulin assembly, facilitating formation of microtubule polymer. In contrast, the molecules and mechanisms that anchor newly formed microtubules at MTOCs are less well characterized. Here we discuss the mechanistic challenges underlying microtubule anchoring, how this is linked with the molecular activities of known and proposed anchoring factors, and what consequences defective microtubule anchoring has at the cellular and organismal level.

Keywords:  MTOC; anchoring; centrosome; microtubule; nucleation

DOI:  https://doi.org/10.3389/fcell.2022.867870
Eur J Pharmacol. 2022 Mar 22. pii: S0014-2999(22)00160-1. [Epub ahead of print] 174899

Multiple actions of NMS-P715, the monopolar spindle 1 (MPS1) mitotic checkpoint inhibitor in liver fluke-associated cholangiocarcinoma cells.

Orawan Waenphimai, Panupong Mahalapbutr, Kulthida Vaeteewoottacharn, Sopit Wongkham, Kanlayanee Sawanyawisuth.

   AIM: NMS-P715 is a potent inhibitor of monopolar spindle 1 (MPS1) mitotic checkpoint kinase. Overexpression of MPS1 is associated with short survival times in patients with cholangiocarcinoma (CCA). This study investigated the anti-cancer effects of NMS-P715 in human CCA cell lines.
MAIN METHODS: KKU-100 and KKU-213A CCA cell lines were treated with NMS-P715 and cell viability was determined using MTT and colony formation assays. Inhibitory effects of NMS-P715 on cell cycle and apoptosis were evaluated using flow cytometry. Expression of underlying mechanism-related proteins was examined by Western blotting. Mitotic catastrophe was assessed by counting abnormal nuclei. Transwell assays were used to examine cell migration and invasion.
KEY FINDINGS: Molecular docking showed that the NMS-P715/MPS1 complex was driven by an induced-fit mechanism. We provide new evidence that NMS-P715 potently inhibited cell proliferation and colony formation in both CCA cell lines. This was accompanied by induction of G2/M arrest and the consequent induction of mitotic catastrophe, a process that occurs during defective mitosis. The recent study showed that NMS-P715 activated caspase-dependent apoptosis and autophagosome formation with an increase of LC3 A/B-II protein expression in CCA cell lines. NMS-P715 also greatly impeded cell migration and invasion in CCA cell lines. The combination of NMS-P715 and gemcitabine or cisplatin showed synergistic effects on CCA cell proliferation.
SIGNIFICANCE: This study revealed for the first time that NMS-P715 is a promising candidate for combating CCA owing via multiple actions and may be suitable for further development in a clinical study.

Keywords:  Apoptosis; Cholangiocarcinoma; MPS1; Mitotic catastrophe; NMS-P715

DOI:  https://doi.org/10.1016/j.ejphar.2022.174899
Fundam Clin Pharmacol. 2022 Mar 26.

Role of NIMA-related Kinase 2 (NEK2) in lung cancer: Mechanisms and therapeutic prospects.

Darshak Shah, Mit Joshi, Bhoomika M Patel.

  The second most common cancer in both males and females is lung cancer. Chemotherapeutic resistance is the main problem associated with the treatment of lung cancer. Radiation therapy and surgery also produce recurrence in lung cancer patients; this shows the need to develop novel agents acting on new targets. A Never in Mitosis (NIMA) Related Kinase 2 (NEK2) is a serine/threonine kinase associated with the family of NIMA-related kinase (NEK). NEK2 plays an important role in the regulating mitotic processes, such as centrosome duplication and separation, kinetochore attachment, spindle assembly checkpoint, and microtubule stabilization. Several in vitro, in vivo, and clinical studies have confirmed the overexpression of NEK2 in various types of cancers including lung cancer. Overexpression of NEK2 in NSCLC cells increased cell proliferation and chromosomal instability. The overexpression of NEK2 results in the activation of its downstream proteins such as β-catenin, MAD2, Hec1, Rootletin, C-Nap1, CDC20, Cep68, and Sgo1. Activation of the Akt, β-catenin, and Wnt pathways could promote growth and metastasis of lung cancer cells. Such confirmation suggests that NEK2 is a novel target for treating many cancers including lung cancer. The current review provides an idea about functions and regulation of NEK2, and emphasizes about the role of NEK2 in lung cancer by discussing in vitro, in vivo and clinical studies pertaining to the same.

Keywords:  NIMA-related Kinase 2 (NEK2); cell cycle; centrosome dysjunction; chromosomal instability; lung cancer; spindle assembly

DOI:  https://doi.org/10.1111/fcp.12777
PLoS One. 2022 ;17(3): e0265692

Profilin is involved in G1 to S phase progression and mitotic spindle orientation during Leishmania donovani cell division cycle.

Bindu Ambaru, Ganesh Muthu Gangadharan, Hosahalli S Subramanya, Chhitar M Gupta.

Profilin is a multi-ligand binding protein, which is a key regulator of actin dynamics and involved in regulating several cellular functions. It is present in all eukaryotes, including trypanosomatids such as Leishmania. However, not much is known about its functions in these organisms. Our earlier studies have shown that Leishmania parasites express a single homologue of profilin (LdPfn) that binds actin, phosphoinositides and poly- L- proline motives, and depletion of its intracellular pool to 50%of normal levels affects the cell growth and intracellular trafficking. Here, we show, employing affinity pull-down and mass spectroscopy, that LdPfn interacted with a large number of proteins, including those involved in mRNA processing and protein translation initiation, such as eIF4A1. Further, we reveal, using mRNA Seq analysis, that depletion of LdPfn in Leishmania cells (LdPfn+/-) resulted in significantly reduced expression of genes which encode proteins involved in cell cycle regulation, mRNA translation initiation, nucleosides and amino acids transport. In addition, we show that in LdPfn+/- cells, cellular levels of eIF4A1 protein were significantly decreased, and during their cell division cycle, G1-to-S phase progression was delayed and orientation of mitotic spindle altered. These changes were, however, reversed to normal by episomal expression of GFP-LdPfn in LdPfn+/- cells. Taken together, our results indicate that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation in Leishmania cell cycle, perhaps through its interaction with elF4A1 protein.

DOI: https://doi.org/10.1371/journal.pone.0265692
Front Cell Dev Biol. 2022 ;10 817831

Phosphorylation of MAD2 at Ser195 Promotes Spindle Checkpoint Defects and Sensitizes Cancer Cells to Radiotherapy in ATM Deficient Cells.

Yang Wang, Tianyu Yu, Yi Han, Yazhi He, Yiran Song, Leiming Guo, Liwei An, Chunying Yang, Feng Wang.

  The spindle assembly checkpoint (SAC) is a critical monitoring device in mitosis for the maintenance of genomic stability. Specifically, the SAC complex comprises several factors, including Mad1, Mad2, and Bub1. Ataxia-telangiectasia mutated (ATM) kinase, the crucial regulator in DNA damage response (DDR), also plays a critical role in mitosis by regulating Mad1 dimerization and SAC. Here, we further demonstrated that ATM negatively regulates the phosphorylation of Mad2, another critical component of the SAC, which is also involved in DDR. Mechanistically, we found that phosphorylation of Mad2 is aberrantly increased in ATM-deficient cells. Point-mutation analysis further revealed that Serine 195 mainly mediated Mad2 phosphorylation upon ATM ablation. Functionally, the phosphorylation of Mad2 causes decreased DNA damage repair capacity and is related to the resistance to cancer cell radiotherapy. Altogether, this study unveils the key regulatory role of Mad2 phosphorylation in checkpoint defects and DNA damage repair in ATM-deficient cells.

Keywords:  ATM kinase; DNA damage repair; checkpoint defect; mad2; phosphorylation

DOI:  https://doi.org/10.3389/fcell.2022.817831
Mol Cell. 2022 Mar 17. pii: S1097-2765(22)00206-4. [Epub ahead of print]

CENP-B-mediated DNA loops regulate activity and stability of human centromeres.

Florian Chardon, Aleksandre Japaridze, Hannes Witt, Leonid Velikovsky, Camellia Chakraborty, Therese Wilhelm, Marie Dumont, Wayne Yang, Carlos Kikuti, Stephane Gangnard, Anne-Sophie Mace, Gijs Wuite, Cees Dekker, Daniele Fachinetti.

  Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

Keywords:  AFM microscopy; CENP; DNA breaks; DNA compaction; DNA topology; centromere; chromosomes; genome stability; optical tweezers; secondary structures

DOI:  https://doi.org/10.1016/j.molcel.2022.02.032
Curr Mol Med. 2022 Mar 21.

Current progress and perspectives of CDC20 in female reproductive cancers.

Ke Ni, Li Hong.

  The cancers of cervix, endometrium, ovary and breast are great threats to women's health. Cancer is characterized by the uncontrolled proliferation of cells and deregulated cell cycle progression is one of the main causes of malignancy. Agents targeting cell cycle regulators may have potential anti-tumor effects. CDC20 (cell division cycle 20 homologue) is a co-activator of the anaphase prompting complex/cyclosome (APC/C) and thus acts as a mitotic regulator. In addition, CDC20 serves as a subunit of the mitotic checkpoint complex (MCC) whose function is to inhibit APC/C. Recently, higher expression of CDC20 was reported in these cancers and was closely associated with their clinicopathological parameters, indicating CDC20 a potential target for cancer treatment that is worth to be further studied. In the present review, we summarized current progress and put forward perspectives of CDC20 in female reproductive cancers.

Keywords:  Breast cancer; CDC20; Cervical cancer; Endometrial cancer; Ovarian cancer

DOI:  https://doi.org/10.2174/1573405618666220321130102
PLoS Genet. 2022 Mar 25. 18(3): e1009860

SUMO-mediated recruitment allows timely function of the Yen1 nuclease in mitotic cells.

Hugo Dorison, Ibtissam Talhaoui, Gerard Mazón.

The post-translational modification of DNA damage response proteins with SUMO is an important mechanism to orchestrate a timely and orderly recruitment of repair factors to damage sites. After DNA replication stress and double-strand break formation, a number of repair factors are SUMOylated and interact with other SUMOylated factors, including the Yen1 nuclease. Yen1 plays a critical role in ensuring genome stability and unperturbed chromosome segregation by removing covalently linked DNA intermediates between sister chromatids that are formed by homologous recombination. Here we show how this important role of Yen1 depends on interactions mediated by non-covalent binding to SUMOylated partners. Mutations in the motifs that allow SUMO-mediated recruitment of Yen1 impair its ability to resolve DNA intermediates and result in chromosome mis-segregation and increased genome instability.

DOI: https://doi.org/10.1371/journal.pgen.1009860
Invest New Drugs. 2022 Mar 21.

The kinesin Eg5 inhibitor K858 exerts antiproliferative and proapoptotic effects and attenuates the invasive potential of head and neck squamous carcinoma cells.

Alice Nicolai, Samanta Taurone, Simone Carradori, Marco Artico, Antonio Greco, Roberta Costi, Susanna Scarpa.

  Our group recently demonstrated that K858, an inhibitor of motor kinesin Eg5, has important antiproliferative and apoptotic effects on breast cancer, prostatic cancer, melanoma and glioblastoma cells. Since high levels of kinesin Eg5 expression have been correlated with a poor prognosis in laryngeal carcinoma, we decided to test the anticancer activity of K858 toward this tumor, which belongs to the group of head and neck squamous cell carcinomas (HNSCCs). These cancers are characterized by low responsiveness to therapy. The effects of K858 on the proliferation and assembly of mitotic spindles of three human HNSCC cell lines were studied using cytotoxicity assays and immunofluorescence for tubulin. The effect of K858 on the cell cycle was analyzed by FACS. The expression levels of cyclin B1 and several markers of apoptosis and invasion were studied by Western blot. Finally, the negative regulation of the malignant phenotype by K858 was evaluated by an invasion assay. K858 inhibited cell replication by rendering cells incapable of developing normal bipolar mitotic spindles. At the same time, K858 blocked the cell cycle in the G2 phase and induced the accumulation of cytoplasmic cyclin B and, eventually, apoptosis. Additionally, K858 inhibited cell migration and attenuated the malignant phenotype. The data described confirm that kinesin Eg5 is an interesting target for new anticancer strategies and suggest that this compound may be a powerful tool for an alternative therapeutic approach to HNSCCs.

Keywords:  Head and neck cancer; K858; Kinesin Eg5; Kinesin KIF11

DOI:  https://doi.org/10.1007/s10637-022-01238-2
Bioorg Chem. 2022 Mar 11. pii: S0045-2068(22)00105-5. [Epub ahead of print]122 105700

Pharmacophore optimization of imidazole chalcones to modulate microtubule dynamics.

Endri Karaj, Samkeliso Dlamini, Radhika Koranne, Shaimaa H Sindi, Lalith Perera, William R Taylor, L M Viranga Tillekeratne.

  We recently reported a new class of imidazole-based chalcones as potential antimitotic agents. In view of their promising cytotoxic activity, a comprehensive structure-activity relationship (SAR) of these compounds was undertaken focusing on four major structural variations: the length of the molecule, the Michael acceptor character, the nature and substitution pattern of ring B, and the nature of the amide functionality tethering ring B. These second-generation analogs (IBCs) demonstrated a superior bioactivity profile than the previously reported imidazole chalcones (referred to as IPEs). The analog IBC-2 with one less methylene group (nor series) and para-fluoro substituted ring B demonstrated the best cytotoxicity profile among the library of compounds. A computational analysis of the NCI-60 data associated both IBCs and the previously reported IPEs with the privileged pharmacological pharmacophore of chalcones. Interestingly, biological studies suggest that the imidazole ring is essential for cytotoxic activity of the elongated chalcone analogues. Immunofluorescence studies revealed that IBC-2, unlike IPEs, has the ability to induce microtubule catastrophe independently of Aurora-B inhibition. The effects of IBC-2 on microtubule dynamics are similar to those of Nocodazole, but the cell cycle effects appear to be different. In-silico studies demonstrate that the members of the new series have the ability to bind to the colchicine binding site of β-tubulin with binding scores similar to those of IPEs, corresponding chalcones and Nocodazole. Although tubulin binding can partially explain the biological effects of IBC-2, on-going target identification studies are aimed at further investigation of its biological targets.

Keywords:  Antimitotic agents; Imidazole chalcones; Microtubule dynamics; Mitotic spindle; SAR

DOI:  https://doi.org/10.1016/j.bioorg.2022.105700
Leuk Res. 2022 Mar 12. pii: S0145-2126(22)00052-2. [Epub ahead of print]116 106826

Midkine inhibitor (iMDK) induces apoptosis of primary effusion lymphoma via G2/M cell cycle arrest.

Mikinori Ueno, Ryusho Kariya, Sittithumcharee Gunya, Kodcharat Cheevapruk, Seiji Okada.

  Primary effusion lymphoma (PEL) is an aggressive B-cell non-Hodgkin lymphoma in immunocompromised individuals such as AIDS patients. PEL shows a poor prognosis (median survival time < 6 months) compared with other AIDS-related lymphomas, and is generally resistant to conventional treatments. Novel drugs for PEL treatment are required. Midkine inhibitor (iMDK) was previously found to suppress midkine protein expression. Interestingly, iMDK suppressed cell proliferation in PEL cell lines in a time- and dose-dependent manner, regardless of midkine gene expression. We examined the mechanism of iMDK on PEL. Importantly, iMDK strongly induced cell cycle arrest at the G2/M phase within 12 h of incubation and suppressed the p-CDK1 protein level, which is associated with the cell cycle checkpoint at G2/M, resulting in mitotic catastrophe with observation of multipolar division. After mitotic catastrophe, iMDK-treated PEL showed apoptosis with caspase-3, - 8, and - 9 activation at 24 h incubation. However, iMDK showed no effects on viral protein-activated signaling pathways such as JAK-STAT, PI3K-Akt and NF-κB, and HHV-8/KSHV gene expression in PEL. These results indicate that iMDK is a novel CDK1 inhibitor and a promising lead compound for PEL chemotherapy treatment.

Keywords:  AIDS; Apoptosis; Cell cycle; Midkine inhibitor (iMDK); Primary effusion lymphoma (PEL)

DOI:  https://doi.org/10.1016/j.leukres.2022.106826
J Phys Chem B. 2022 Mar 22.

Selective Recognition of Phosphatidylinositol Phosphate Receptors by C-Terminal Tail of Mitotic Kinesin-like Protein 2 (MKlp2).

Hyunhyuk Tae, Soohyun Park, Seong-Oh Kim, Saziye Yorulmaz Avsar, Nam-Joon Cho.

The mitotic kinesin-like protein 2 (MKlp2) plays a key role in the proper completion of cytokinetic abscission. Specifically, the C-terminal tail of MKlp2 (CTM peptides) offers a stable tethering on the plasma membrane and microtubule cytoskeleton in the midbody during abscission. However, little is known about the underlying mechanism of how the CTM peptides bind to the plasma membrane of the intercellular bridge. Herein, we identify the specific molecular interaction between the CTM peptides and phosphatidylinositol phosphate (PIP) receptors using quartz crystal microbalance-dissipation and atomic force microscopy force spectroscopic measurements. To systematically examine the effects of amino acids, we designed a series of synthetic 33-mer peptides derived from the wild-type (CTM1). First, we evaluated the peptide binding amount caused by electrostatic interactions based on 100% zwitterionic and 30% negatively charged model membranes, whereby the nonspecific attractions were nearly proportional to the net charge of peptides. Upon incubating with PIP-containing model membranes, the wild-type CTM1 and its truncated mutation showed significant PI(3)P-specific binding, which was evidenced by a 15-fold higher binding mass and 6-fold stronger adhesion force compared to other negatively charged membranes. The extent of the specific binding was predominantly dependent on the existence of S21, whereby substitution or deletion of S21 significantly hindered the binding affinity. Taken together, our findings based on a correlative measurement platform enabled the quantification of the nonelectrostatic, selective binding interactions of the C-terminal of MKlp2 to certain PIP receptors and contributed to understanding the molecular mechanisms on complete cytokinetic abscission in cells.

DOI: https://doi.org/10.1021/acs.jpcb.1c10534
Rejuvenation Res. 2022 Mar 22.

Leveraging substrate stiffness to promote stem cell asymmetric division via mechanotransduction-polarity protein axis and its Bayesian regression analysis.

Ankita Das, Shreya Adhikary, Amit Roy Chowdhury, Ananya Barui.

Asymmetric division of stem cells is an evolutionarily conserved process in multicellular organisms responsible for maintaining cellular fate diversity. Symmetric-asymmetric division pattern of mesenchymal stem cells (MSC) is regulated by both biochemical and biophysical cues. However, modulation of mechanotransduction pathway by varying scaffold properties and their adaptation to control stem cell division fate is not widely established. In present study, we explored the interplay between the mechanotrasduction pathway and polarity protein complex in stem cell asymmetry under varied biophysical stimuli. We hypothesize that variation of scaffold stiffness will impart mechanical stimulus and control the cytoskeleton assembly through RhoA, which will lead to further downstream activation of polarity-related cell signalling and asymmetric division of MSC. To establish the hypothesis, umbilical cord derived MSC were cultured on PCL/collagen scaffolds with varied stiffness and expressions of several important genes (viz. YAP, TAZ, LATS1, LATS2, Par3, Par6, PRKC1 (homolog of aPKC) and RhoA) and biomarkers (viz. YAP, TAZ, F-actin, Numb) were assessed. SVM polarity index was employed to understand the polarization status of the MSC cultured on varied scaffold stiffness. Further, the Bayesian logistic regression model was employed for classifying the asymmetric division of MSC cultured on different scaffold stiffness which showed 91% accuracy. Present study emphasizes the vital role of scaffold properties in modulating the mechanotransduction signalling pathway of MSC and provides mechanistic basis for adopting facile method to control stem cell division pattern towards improving tissue engineering outcome.

DOI: https://doi.org/10.1089/rej.2021.0039