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Title: Salivary protein histatin 3 regulates cell proliferation by enhancing p27{sup Kip1} and heat shock cognate protein 70 ubiquitination

Abstract

Histatins are salivary proteins with antimicrobial activities. We previously reported that histatin 3 binds to heat shock cognate protein 70 (HSC70), which is constitutively expressed, and induces DNA synthesis stimulation and promotes human gingival fibroblast (HGF) survival. However, the underlying mechanisms of histatin 3 remain largely unknown. Here, we found that the KRHH sequence of histatin 3 at the amino acid positions 5–8 was essential for enhancing p27{sup Kip1} (a cyclin-dependent kinase inhibitor) binding to HSC70 that occurred in a dose-dependent manner; histatin 3 enhanced the binding between p27{sup Kip1} and HSC70 during the G{sub 1}/S transition of HGFs as opposed to histatin 3-M(5–8) (substitution of KRHH for EEDD in histatin 3). Histatin 3, but not histatin 3-M(5–8), stimulated DNA synthesis and promoted HGF survival. Histatin 3 dose-dependently enhanced both p27{sup Kip1} and HSC70 ubiquitination, whereas histatin 3-M(5–8) did not. These findings provide further evidence that histatin 3 may be involved in the regulation of cell proliferation, particularly during G{sub 1}/S transition, via the ubiquitin–proteasome system of p27{sup Kip1} and HSC70. - Highlights: • KRHH amino acid sequence was required in histatin 3 to bind HSC70. • Histatin 3 enhanced HSC70 binding to p27{sup Kip1} during the G{sub 1}/S transitionmore » in HGFs. • KRHH sequence stimulated DNA synthesis and promoted cell survival. • Histatin 3 dose-dependently enhanced both p27{sup Kip1} and HSC70 ubiquitination. • Histatin 3 stimulates cell proliferation via the ubiquitin–proteasome system.« less

Authors:
 [1];  [2];  [3];  [1]
  1. Department of Pharmacology, Matsumoto Dental University, Shiojiri, Nagano 399-0781 (Japan)
  2. Department of Bacteriology, Osaka Dental University, Hirakata, Osaka 573-1121 (Japan)
  3. Department of Dental Education Innovation, Osaka Dental University, Hirakata, Osaka 573-1121 (Japan)
Publication Date:
OSTI Identifier:
22594218
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 470; Journal Issue: 2; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMINO ACID SEQUENCE; AMINO ACIDS; BROMIDES; CATTLE; CELL PROLIFERATION; DNA; FIBROBLASTS; FLUORESCENCE; GLUTATHIONE; HEAT; HEAT-SHOCK PROTEINS; KIDNEYS; NEOPLASMS; TRANSFERASES

Citation Formats

Imamura, Yasuhiro, E-mail: yimamura@po.mdu.ac.jp, Wang, Pao-Li, Masuno, Kazuya, and Sogawa, Norio. Salivary protein histatin 3 regulates cell proliferation by enhancing p27{sup Kip1} and heat shock cognate protein 70 ubiquitination. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.01.072.
Imamura, Yasuhiro, E-mail: yimamura@po.mdu.ac.jp, Wang, Pao-Li, Masuno, Kazuya, & Sogawa, Norio. Salivary protein histatin 3 regulates cell proliferation by enhancing p27{sup Kip1} and heat shock cognate protein 70 ubiquitination. United States. doi:10.1016/J.BBRC.2016.01.072.
Imamura, Yasuhiro, E-mail: yimamura@po.mdu.ac.jp, Wang, Pao-Li, Masuno, Kazuya, and Sogawa, Norio. Fri . "Salivary protein histatin 3 regulates cell proliferation by enhancing p27{sup Kip1} and heat shock cognate protein 70 ubiquitination". United States. doi:10.1016/J.BBRC.2016.01.072.
@article{osti_22594218,
title = {Salivary protein histatin 3 regulates cell proliferation by enhancing p27{sup Kip1} and heat shock cognate protein 70 ubiquitination},
author = {Imamura, Yasuhiro, E-mail: yimamura@po.mdu.ac.jp and Wang, Pao-Li and Masuno, Kazuya and Sogawa, Norio},
abstractNote = {Histatins are salivary proteins with antimicrobial activities. We previously reported that histatin 3 binds to heat shock cognate protein 70 (HSC70), which is constitutively expressed, and induces DNA synthesis stimulation and promotes human gingival fibroblast (HGF) survival. However, the underlying mechanisms of histatin 3 remain largely unknown. Here, we found that the KRHH sequence of histatin 3 at the amino acid positions 5–8 was essential for enhancing p27{sup Kip1} (a cyclin-dependent kinase inhibitor) binding to HSC70 that occurred in a dose-dependent manner; histatin 3 enhanced the binding between p27{sup Kip1} and HSC70 during the G{sub 1}/S transition of HGFs as opposed to histatin 3-M(5–8) (substitution of KRHH for EEDD in histatin 3). Histatin 3, but not histatin 3-M(5–8), stimulated DNA synthesis and promoted HGF survival. Histatin 3 dose-dependently enhanced both p27{sup Kip1} and HSC70 ubiquitination, whereas histatin 3-M(5–8) did not. These findings provide further evidence that histatin 3 may be involved in the regulation of cell proliferation, particularly during G{sub 1}/S transition, via the ubiquitin–proteasome system of p27{sup Kip1} and HSC70. - Highlights: • KRHH amino acid sequence was required in histatin 3 to bind HSC70. • Histatin 3 enhanced HSC70 binding to p27{sup Kip1} during the G{sub 1}/S transition in HGFs. • KRHH sequence stimulated DNA synthesis and promoted cell survival. • Histatin 3 dose-dependently enhanced both p27{sup Kip1} and HSC70 ubiquitination. • Histatin 3 stimulates cell proliferation via the ubiquitin–proteasome system.},
doi = {10.1016/J.BBRC.2016.01.072},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 470,
place = {United States},
year = {Fri Feb 05 00:00:00 EST 2016},
month = {Fri Feb 05 00:00:00 EST 2016}
}
  • Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found tomore » be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.« less
  • The movement protein (MP) of bipartite geminiviruses facilitates cell-to-cell as well as long-distance transport within plants and influences viral pathogenicity. Yeast two-hybrid assays identified a chaperone, the nuclear-encoded and plastid-targeted heat shock cognate 70 kDa protein (cpHSC70-1) of Arabidopsis thaliana, as a potential binding partner for the Abutilon mosaic virus (AbMV) MP. In planta, bimolecular fluorescence complementation (BiFC) analysis showed cpHSC70-1/MP complexes and MP homooligomers at the cell periphery and co-localized with chloroplasts. BiFC revealed cpHSC70-1 oligomers associated with chloroplasts, but also distributed at the cellular margin and in filaments arising from plastids reminiscent of stromules. Silencing the cpHSC70 genemore » of Nicotiana benthamiana using an AbMV DNA A-derived gene silencing vector induced minute white leaf areas, which indicate an effect on chloroplast stability. Although AbMV DNA accumulated within chlorotic spots, a spatial restriction of these occurred, suggesting a functional relevance of the MP-chaperone interaction for viral transport and symptom induction.« less
  • In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27 Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groupsmore » of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less
  • The cyclin-dependent kinase (CDK) inhibitor p27{sup Kip1} is an important regulator of cell cycle progression as it negatively regulates G{sub 0/1} progression and plays a major role in controlling the cell cycle. The screening of the p27{sup Kip1} sequence identified many potential phosphorylation sites. Although Ser{sup 1} and Thr{sup 187} were shown to be important for p27{sup Kip1} function, the effects of a combined deletion of both sites on p27{sup Kip1} function are still unknown. To investigate the effects of the overexpression of exogenous p27{sup Kip1} protein lacking both the Ser{sup 1} and Thr{sup 187} sites on subcellular localization, cellmore » cycle, and proliferation, a plasmid was constructed containing mutations of p27{sup Kip1} at Ser{sup 1} and Thr{sup 187} (S10A/T187A p27), and transfected into the HepG{sub 2} cell line with Lipofectamine. Wild-type and mutant p27 plasmids S10A and T187A were transfected separately as control groups. As a result, the proliferation of HepG{sub 2} cells was greatly inhibited and cell cycle was arrested in G{sub 0/1} phase after exogenous p27{sup Kip1} double-mutant expression. All recombinant p27{sup Kip1} constructs were distributed in the nucleus after synchronization in G phase by treatment with leptomycin B. The expressed wild-type and T187A p27{sup Kip1} proteins were translocated from the nucleus into cytoplasm when cells were exposed to 20% serum for 8 h, whereas the S10A p27{sup Kip1} and S10A/T187A p27{sup Kip1} proteins remained in the nucleus. FACS profiles and cell growth curves indicated that the Ser{sup 1} and Thr{sup 187} double mutant has no significant effect on the biological activities of cell cycle control and growth inhibition. Our results suggest that expression of the p27{sup Kip1} double-mutant abolishes its cytoplasmic redistribution but does not abrogate G{sub 0/1} phase arrest in the HepG{sub 2} cell line.« less
  • Highlights: •Expression of p21 and p27 in the hearts showed a peak during postnatal stages. •p21 and p27 bound to cyclin E, cyclin A and CDK2 in the hearts at postnatal stages. •Cardiomyocytes in both KO mice showed failure in the cell cycle exit at G1-phase. •These data show the first apparent phenotypes in the hearts of Cip/Kip KO mice. -- Abstract: Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remainsmore » largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21{sup Cip1} and p27{sup Kip1} but not p57{sup Kip2} showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21{sup Cip1} and p27{sup Kip1} bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21{sup Cip1} and p27{sup Kip1} knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21{sup Cip1} and p27{sup Kip} play important roles in the cell cycle exit of postnatal cardiomyocytes.« less