scholarly journals Characterization of a novel separase-interacting protein and candidate new securin, Eip1p, in the fungal pathogen Candida albicans

2019 ◽  
Vol 30 (19) ◽  
pp. 2469-2489 ◽  
Author(s):  
Samantha Sparapani ◽  
Catherine Bachewich
Keyword(s):  
Candida Albicans ◽  
Chromosome Segregation ◽  
Affinity Purification ◽  
Sequence Divergence ◽  
Dependent Manner ◽  
Uncharacterized Protein ◽  
Interacting Protein ◽  
Functional Analyses ◽  
Anti Fungal ◽  
Mitotic Regulation

Proper chromosome segregation is crucial for maintaining genomic stability and dependent on separase, a conserved and essential cohesin protease. Securins are key regulators of separases, but remain elusive in many organisms due to sequence divergence. Here, we demonstrate that the separase homologue Esp1p in the ascomycete Candida albicans, an important pathogen of humans, is essential for chromosome segregation . However, C. albicans lacks a sequence homologue of securins found in model ascomycetes. We sought a functional homologue through identifying Esp1p interacting factors. Affinity purification of Esp1p and mass spectrometry revealed Esp1p-Interacting Protein1 (Eip1p)/Orf19.955p, an uncharacterized protein specific to Candida species. Functional analyses demonstrated that Eip1p is important for chromosome segregation but not essential, and modulated in an APCCdc20-dependent manner, similar to securins. Eip1p is strongly enriched in response to methyl methanesulfate (MMS) or hydroxyurea (HU) treatment, and its depletion partially suppresses an MMS or HU-induced metaphase block. Further, Eip1p depletion reduces Mcd1p/Scc1p, a cohesin subunit and separase target. Thus, Eip1p may function as a securin. However, other defects in Eip1p-depleted cells suggest additional roles. Overall, the results introduce a candidate new securin, provide an approach for identifying these divergent proteins, reveal a putative anti-fungal therapeutic target, and highlight variations in mitotic regulation in eukaryotes.

scholarly journals Aurora kinase Ipl1 facilitates bilobed distribution of clustered kinetochores to ensure error-free chromosome segregation in Candida albicans

2019 ◽  
Author(s):  
Neha Varshney ◽  
Kaustuv Sanyal
Keyword(s):  
Candida Albicans ◽  
Chromosome Segregation ◽  
Structural Integrity ◽  
Aurora Kinase ◽  
Spindle Pole ◽  
Microtubule Dynamics ◽  
Aurora Kinase B ◽  
Spindle Pole Bodies ◽  
Candida Infections ◽  
Anti Fungal

Candida albicans, an ascomycete, has an ability to switch to diverse morphological forms. While C. albicans is predominatly diploid, it can tolerate aneuploidy as a survival strategy under stress. Aurora kinase B homolog Ipl1 is a critical ploidy regulator that controls microtubule dynamics and chromosome segregation in Saccharomyces cerevisiae. In this study, we show that Ipl1 in C. albicans has a longer activation loop than that of the well-studied ascomycete S. cerevisiae. Ipl1 localizes to the kinetochores during the G1/S phase and associates with the spindle during mitosis. Ipl1 regulates cell morphogenesis and is required for cell viability. Ipl1 monitors microtubule dynamics which is mediated by separation of spindle pole bodies. While Ipl1 is dispensable for maintaining structural integrity and clustering of kinetochores in C. albicans, it is required for the maintenance of kinetochore geometry to form bilobed structures along the mitotic spindle, a feature of Ipl1 that was not observed in other yeasts. Depletion of Ipl1 results in erroneous kinetochore-microtubule attachments leading to aneuploidy-associated resistance to fluconazole, the most common anti-fungal drug used to treat Candida infections. Taking together, we suggest that Ipl1 spatiotemporally ensures kinetochore geometry to facilitate bipolar spindle assembly crucial for ploidy maintenance in C. albicans.

scholarly journals Cdh1 Regulates Cell Cycle through Modulating the Claspin/Chk1 and the Rb/E2F1 Pathways

2009 ◽  
Vol 20 (14) ◽  
pp. 3305-3316 ◽  
Author(s):  
Daming Gao ◽  
Hiroyuki Inuzuka ◽  
Michael Korenjak ◽  
Alan Tseng ◽  
Tao Wu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressors ◽  
Affinity Purification ◽  
Human Fibroblasts ◽  
Premature Senescence ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Damage Repair ◽  
Rb Pathway ◽  
Phase Entry

APC/Cdh1 is a major cell cycle regulator and its function has been implicated in DNA damage repair; however, its exact role remains unclear. Using affinity purification coupled with mass spectrometry, we identified Claspin as a novel Cdh1-interacting protein and further demonstrated that Claspin is a novel Cdh1 ubiquitin substrate. As a result, inactivation of Cdh1 leads to activation of the Claspin/Chk1 pathway. Previously, we demonstrated that Rb interacts with Cdh1 to influence its ability to degrade Skp2. Here, we report that Cdh1 reciprocally regulates the Rb pathway through competing with E2F1 to bind the hypophosphorylated form of Rb. Although inactivation of Cdh1 in HeLa cells, with defective p53/Rb pathways, led to premature S phase entry, acute depletion of Cdh1 in primary human fibroblasts resulted in premature senescence. Acute loss of many other major tumor suppressors, including PTEN and VHL, also induces premature senescence in a p53- or Rb-dependent manner. Similarly, we showed that inactivation of the p53/Rb pathways by overexpression of SV40 LT-antigen partially reversed Cdh1 depletion–induced growth arrest. Therefore, loss of Cdh1 is only beneficial to cells with abnormal p53 and Rb pathways, which helps explain why Cdh1 loss is not frequently found in many tumors.

Efficacy of Novel Schiff base Derivatives as Antifungal Compounds in Combination with Approved Drugs Against Candida Albicans

2019 ◽  
Vol 15 (6) ◽  
pp. 648-658 ◽  
Author(s):  
Manzoor Ahmad Malik ◽  
Shabir Ahmad Lone ◽  
Parveez Gull ◽  
Ovas Ahmad Dar ◽  
Mohmmad Younus Wani ◽  
...  
Keyword(s):  
Schiff Base ◽  
Candida Albicans ◽  
Antifungal Activity ◽  
Fungal Infections ◽  
Total Sterol ◽  
Antifungal Drugs ◽  
New Drugs ◽  
Ergosterol Biosynthesis ◽  
Dependent Manner ◽  
Schiff Base Derivatives

Background: The increasing incidence of fungal infections, especially caused by Candida albicans, and their increasing drug resistance has drastically increased in recent years. Therefore, not only new drugs but also alternative treatment strategies are promptly required. Methods: We previously reported on the synergistic interaction of some azole and non-azole compounds with fluconazole for combination antifungal therapy. In this study, we synthesized some non-azole Schiff-base derivatives and evaluated their antifungal activity profile alone and in combination with the most commonly used antifungal drugs- fluconazole (FLC) and amphotericin B (AmB) against four drug susceptible, three FLC resistant and three AmB resistant clinically isolated Candida albicans strains. To further analyze the mechanism of antifungal action of these compounds, we quantified total sterol contents in FLC-susceptible and resistant C. albicans isolates. Results: A pyrimidine ring-containing derivative SB5 showed the most potent antifungal activity against all the tested strains. After combining these compounds with FLC and AmB, 76% combinations were either synergistic or additive while as the rest of the combinations were indifferent. Interestingly, none of the combinations was antagonistic, either with FLC or AmB. Results interpreted from fractional inhibitory concentration index (FICI) and isobolograms revealed 4-10-fold reduction in MIC values for synergistic combinations. These compounds also inhibit ergosterol biosynthesis in a concentration-dependent manner, supported by the results from docking studies. Conclusion: The results of the studies conducted advocate the potential of these compounds as new antifungal drugs. However, further studies are required to understand the other mechanisms and in vivo efficacy and toxicity of these compounds.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

scholarly journals Necroptosis protects against exacerbation of acute pancreatitis

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Michittra Boonchan ◽  
Hideki Arimochi ◽  
Kunihiro Otsuka ◽  
Tomoko Kobayashi ◽  
Hisanori Uehara ◽  
...  
Keyword(s):  
Acute Pancreatitis ◽  
Necrotizing Pancreatitis ◽  
Neutrophil Infiltration ◽  
Dependent Manner ◽  
Protective Effects ◽  
Interacting Protein ◽  
Inflammatory Conditions ◽  
Edematous Pancreatitis ◽  
Genetic Deletion ◽  
Dose Dependent

AbstractThe sensing of various extrinsic stimuli triggers the receptor-interacting protein kinase-3 (RIPK3)-mediated signaling pathway, which leads to mixed-lineage kinase-like (MLKL) phosphorylation followed by necroptosis. Although necroptosis is a form of cell death and is involved in inflammatory conditions, the roles of necroptosis in acute pancreatitis (AP) remain unclear. In the current study, we administered caerulein to Ripk3- or Mlkl-deficient mice (Ripk3−/− or Mlkl−/− mice, respectively) and assessed the roles of necroptosis in AP. We found that Ripk3−/− mice had significantly more severe pancreatic edema and inflammation associated with macrophage and neutrophil infiltration than control mice. Consistently, Mlkl−/− mice were more susceptible to caerulein-induced AP, which occurred in a time- and dose-dependent manner, than control mice. Mlkl−/− mice exhibit weight loss, edematous pancreatitis, necrotizing pancreatitis, and acinar cell dedifferentiation in response to tissue damage. Genetic deletion of Mlkl resulted in downregulation of the antiapoptotic genes Bclxl and Cflar in association with increases in the numbers of apoptotic cells, as detected by TUNEL assay. These findings suggest that RIPK3 and MLKL-mediated necroptosis exerts protective effects in AP and caution against the use of necroptosis inhibitors for AP treatment.

scholarly journals Structural and dynamic mechanisms of CBF3-guided centromeric nucleosome formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ruifang Guan ◽  
Tengfei Lian ◽  
Bing-Rui Zhou ◽  
Emily He ◽  
Carl Wu ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Chromosome Segregation ◽  
Budding Yeast ◽  
Dna Conformation ◽  
Dependent Manner ◽  
Dynamic Interactions ◽  
Nucleosome Core ◽  
Dynamic Mechanisms ◽  
Nucleosome Formation ◽  
Core Histones

AbstractAccurate chromosome segregation relies on the specific centromeric nucleosome–kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.

scholarly journals Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

2007 ◽  
Vol 6 (6) ◽  
pp. 931-939 ◽  
Author(s):  
Fang Li ◽  
Michael J. Svarovsky ◽  
Amy J. Karlsson ◽  
Joel P. Wagner ◽  
Karen Marchillo ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Adhesion ◽  
Biofilm Formation ◽  
Fungal Infections ◽  
Gene Dosage ◽  
Venous Catheter ◽  
Flow Chamber ◽  
Dependent Manner

ABSTRACT Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.

scholarly journals Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement

1992 ◽  
Vol 284 (1) ◽  
pp. 169-176 ◽  
Author(s):  
T R Hughes ◽  
S J Piddlesden ◽  
J D Williams ◽  
R A Harrison ◽  
B P Morgan
Keyword(s):  
Affinity Purification ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Inhibitory Protein ◽  
Rat Erythrocytes ◽  
Butanol Extract ◽  
Isolation And Characterization ◽  
Membrane Bound

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated ‘reactive’ lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

scholarly journals Identification of Nucleolin as a Novel AEG-1-Interacting Protein in Breast Cancer via Interactome Profiling

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2842
Author(s):  
Seong-Jae Lee ◽  
Kyoung-Min Choi ◽  
Geul Bang ◽  
Seo-Gyu Park ◽  
Eun-Bi Kim ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Cells ◽  
Molecular Mechanisms ◽  
Affinity Purification ◽  
Ectopic Expression ◽  
Cancer Cell Proliferation ◽  
Breast Cancer Cell Proliferation ◽  
Interacting Protein ◽  
Proliferation And Invasion ◽  
Oncogenic Function

Breast cancer is one of the most common malignant diseases worldwide. Astrocyte elevated gene-1 (AEG-1) is upregulated in breast cancer and regulates breast cancer cell proliferation and invasion. However, the molecular mechanisms by which AEG-1 promotes breast cancer have yet to be fully elucidated. In order to delineate the function of AEG-1 in breast cancer development, we mapped the AEG-1 interactome via affinity purification followed by LC-MS/MS. We identified nucleolin (NCL) as a novel AEG-1 interacting protein, and co-immunoprecipitation experiments validated the interaction between AEG-1 and NCL in breast cancer cells. The silencing of NCL markedly reduced not only migration/invasion, but also the proliferation induced by the ectopic expression of AEG-1. Further, we found that the ectopic expression of AEG-1 induced the tyrosine phosphorylation of c-Met, and NCL knockdown markedly reduced this AEG-1 mediated phosphorylation. Taken together, our report identifies NCL as a novel mediator of the oncogenic function of AEG-1, and suggests that c-Met could be associated with the oncogenic function of the AEG-1-NCL complex in the context of breast cancer.

Expression and localization of androgen receptor-interacting protein-4 in the testis

2007 ◽  
Vol 292 (2) ◽  
pp. E513-E522 ◽  
Author(s):  
Andrii Domanskyi ◽  
Fu-Ping Zhang ◽  
Mirja Nurmio ◽  
Jorma J. Palvimo ◽  
Jorma Toppari ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Hormone Receptor ◽  
Luteinizing Hormone Receptor ◽  
Dependent Manner ◽  
Cell Nuclei ◽  
Interacting Protein ◽  
Independent Functions

Androgen receptor-interacting protein 4 (ARIP4) belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA excision repair, and homologous recombination. It is a DNA-dependent ATPase, binds to DNA and mononucleosomes, and interacts with androgen receptor (AR) and modulates AR-dependent transactivation. We have examined in this study the expression and cellular localization of ARIP4 during postnatal development of mouse testis. ARIP4 was detected by immunohistochemistry in Sertoli cell nuclei at all ages studied, starting on day 5, and exhibited the highest expression level in adult mice. At the onset of spermatogenesis, ARIP4 expression became evident in spermatogonia, pachytene, and diplotene spermatocytes. Immunoreactive ARIP4 antigen was present in Leydig cell nuclei. In Sertoli cells ARIP4 was expressed in a stage-dependent manner, with high expression levels at stages II–VI and VII–VIII. ARIP4 expression patterns did not differ significantly in testes of wild-type, follicle-stimulating hormone receptor knockout, and luteinizing hormone receptor knockout mice. In testes of hypogonadal mice, ARIP4 was found mainly in interstitial cells and exhibited lower expression in Sertoli and germ cells. In vitro stimulation of rat seminiferous tubule segments with testosterone, FSH, or forskolin did not significantly change stage-specific levels of ARIP4 mRNA. Heterozygous ARIP4+/− mice were haploinsufficient and had reduced levels of Sertoli-cell specific androgen-regulated Rhox5 (also called Pem) mRNA. Collectively, ARIP4 is an AR coregulator in Sertoli cells in vivo, but the expression in the germ cells implies that it has also AR-independent functions in spermatogenesis.

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