P21 Research & Studies

The purpose of the present study was to prepare novel anti-senescent peptides from pearls, characterize their primary sequence and secondary structure, and investigate their protective effects and molecular mechanisms towards D-galactose (D-gal)-induced senescence on human dermal fibroblasts (HDFs). Novel pearl peptides with a purity of 96.58 % and maximum yield of 3.29 % were obtained using ultrasonic-assisted acetic acid extraction strategy under the optimal extraction conditions (ultrasonic power 200 W, ultrasonic time 70 min, and the ratio of pearl powder to acetic acid 1:20). It is sequenced mainly as five novel anti-senescent peptides with molecular weight < 2000 Da, and consisted of β-sheet (43.2 %), random coil (32.1 %), β-turn (21.2 %) and α-helix (3.5 %) analyzed by LC-MS/MS, FT-IR and CD spectroscopy. Further anti-senescent experiments showed that pearl peptides can increase cell viability, restore DNA damage, and suppress the accumulation of ROS as well as senescence-associated-β-galactosidase (SA-β-gal). The molecular mechanism may be that pearl peptides down-regulate the gene and protein expressions of senescence-associated proteins p53, p21, and p16. Therefore, novel pearl peptides could be developed as functional foods or nutritional supplements for the prevention of skin aging.

Finding the oncogene, which was able to inhibit tumor cells intrinsically and improve the immune answers, will be the future direction for renal cancer combined treatment. Following patient sample analysis and signaling pathway examination, we propose p21-activated kinase 4 (PAK4) as a potential target drug for kidney cancer. PAK4 exhibits high expression levels in patient samples and plays a regulatory role in the immune microenvironment.

LncRNA STK4 antisense RNA 1 (STK4-AS1) has been identified as a potential biomarker associated with multiple cancers. We proposed that STK4-AS1 plays a role in the proliferation of osteosarcoma by regulating the cell cycle.

Rapeseed peptide, extracted from rapeseed protein, is known to have a variety of biological activities. In this study, the anti-proliferation effect and molecular mechanism of rapeseed peptide on HepG2 cells were investigated.

A senescence-associated secretory phenotype (SASP) and a mild inflammatory response characteristic of senescent cells (inflammaging) form the conditions for the development of cardiovascular diseases: atherosclerosis, coronary heart disease, and myocardial infarction. The purpose of the review is to analyze the pool of signaling molecules that form SASP and inflammaging in cells of the cardiovascular system and to search for targets for the action of vasoprotective peptides. The SASP of cells of the cardiovascular system is characterized by a change in the synthesis of anti-proliferative proteins (p16, p19, p21, p38, p53), cytokines characteristic of inflammaging (IL-1α,β, IL-4, IL-6, IL-8, IL-18, TNFα, TGFβ1, NF-κB, MCP), matrix metalloproteinases, adhesion molecules, and sirtuins. It has been established that peptides are physiological regulators of body functions. Vasoprotective polypeptides (liraglutide, atrial natriuretic peptide, mimetics of relaxin, Ucn1, and adropin), KED tripeptide, and AEDR tetrapeptide regulate the synthesis of molecules involved in inflammaging and SASP-forming cells of the cardiovascular system. This indicates the prospects for the development of drugs based on peptides for the treatment of age-associated cardiovascular pathology.

Alzheimer's disease (AD) is a neurodegeneration csharacterized by amyloid-β (Aβ) deposition and abnormally phosphorylated Tau protein aggregation. Autophagy, as an important cellular metabolic activity, is closely related to the production, secretion and clearance of Aβ peptide and Tau phosphorylation level. Therefore, autophagy may become a potential target for AD treatment. A large number of molecules are involved in the mammalian target of rapamycin (mTOR)-dependent or mTOR-independent pathway of autophagy. More and more evidences show that statins can intervene autophagy by regulating the activity or expression level of autophagy-related proteins and genes. On the one hand, statins can induce autophagy through Sirtuin1 (SIRT1), P21, nuclear P53 and adenylate activated protein kinase (AMPK). On the other hand, statins inhibit the mevalonate metabolism pathway, thereby interfering with the prenylation of small GTPases, leading to autophagy dysfunction. Statins can also reduce the levels of LAMP2 and dynein, destroying autophagy. In this review, we focused on the role of autophagy in AD and the autophagy mechanism of statins in the potential treatment of AD.

The oncoprotein binding (OPB) domain of Yin Yang 1 (YY1) consists of 26 amino acids between G201 and S226, and is involved in YY1 interaction with multiple oncogene products, including MDM2, AKT, EZH2 and E1A. Through the OPB domain, YY1 promotes the oncogenic or proliferative regulation of these oncoproteins in cancer cells. We previously demonstrated that a peptide with the OPB sequence blocked YY1-AKT interaction and inhibited breast cancer cell proliferation.

Cardiac hypertrophy increases the risk of morbidity and mortality of cardiovascular disease and thus inhibiting such hypertrophy is beneficial. In the present study, we explored the effect of a bioactive peptide (PAP) on angiotensin II (Ang II)-induced hypertrophy and associated ventricular arrhythmias in in vitro and in vivo models. PAP enhances p21 activated kinase 1 (Pak1) activity by increasing the level of phosphorylated Pak1 in cultured neonatal rat ventricular myocytes (NRVMs). Such PAP-induced Pak1 activation is associated with a significant reduction of Ang II-induced hypertrophy in NRVMs and C57BL/6 mice, in vitro and in vivo, respectively. Furthermore, PAP antagonizes ventricular arrhythmias associated with Ang II-induced hypertrophy in mice. Its antiarrhythmic effect is likely to be involved in multiple mechanisms to affect both substrate and trigger of ventricular arrhythmogenesis. Thus our results suggest that Pak1 activation achieved by specific bioactive peptide represents a potential novel therapeutic strategy for cardiac hypertrophy and associated ventricular arrhythmias.

We have employed computer-based molecular modeling approaches to design peptides from the ras-p21 and p53 proteins that either induce tumor cell reversion to the untransformed phenotype or induce tumor cell necrosis without affecting normal cells. For rasp21, we have computed and superimposed the average low energy structures for the wild-type protein and oncogenic forms of this protein and found that specific domains change conformation in the oncogenic proteins. We have synthesized peptides corresponding to these and found that ras peptides, 35-47 (PNC-7) and 96-110 (PNC-2), block oncogenic ras-p21-induced oocyte maturation but have no effect on insulin-induced oocyte maturation that requires activation of endogenous wild-type ras-p21. These results show signal transduction pathway differences between oncogenic and activated wild-type ras-p21. Both peptides, attached to a membrane-penetrating peptide (membrane residency peptide or MRP), either induce phenotypic reversion to the untransformed phenotype or tumor cell necrosis of several ras-transformed cell lines, but have no effect on the growth of normal cells. Using other computational methods, we have designed two peptides, PNC-27 and 28, containing HDM-2-protein-binding domain sequences from p53 linked on their C-termini to the MRP that induce pore formation in the membranes of a wide range of cancer cells but not any normal cells tested. This is due to the expression of HDM-2 in the cancer cell membrane that does not occur in normal cells. These peptides eradicate a highly malignant tumor in nude mice with no apparent side effects. Both ras and p53 peptides show promise as anti-tumor agents in humans.

To evaluate the internalization and nuclear translocation of (123)I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1).

Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.

Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH(2) terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.

The tumour suppressor protein p21(WAF1) plays a central role in regulating eukaryotic cell-cycle progression. Through its association with G1- and S-phase CDK complexes it regulates activation of the retinoblastoma protein (pRb) and E2F transcription factors. Recognition of CDK/cyclin complexes by p21 occurs, at least in part, through a protein-protein interaction with a binding groove on the cyclin subunit. The same groove has been shown to be involved in the recruitment of macromolecular CDK substrates, including pRb and E2F. Blocking of this recruitment site therefore prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of p21-like tumour suppression. Starting from the C-terminal cyclin-binding domain of p21 we have identified the minimal and optimized bioactive (152)HAKRRLIF(159) peptide sequence with respect to CDK protein kinase inhibition where pRb is the substrate. The phosphorylation of histone H1, however, which does not contain a recognizable cyclin-binding motif, was unaffected. Detailed structure-activity relationship investigations revealed that the determinants within this sequence are residues Arg(155), Leu(157) and Phe(159) and more completely define the composition of the cyclin-binding motif. A marked increase in potency was obtained upon replacement of the native Ser(153) with an Ala residue in the context of short synthetic peptide inhibitors and significantly, this mutation resulted in comparable affinity with CDK2/cyclin A as does the full-length recombinant p21 (which has CDK2 and cyclin A binding sites). Peptides derived from various proteins known to interact with cyclins were compared for potency and selectivity. A molecular model of the complex between the cyclin groove and the HAKRRLIF peptide was constructed. This model accounts for the observed peptide structure-activity relationships, including the potency enhancement of the LIF sequence occupying the hydrophobic pocket. Furthermore, it provides generic insights into molecular interactions governing cyclin groove recognition and lays the foundation for the development of peptidomimetic inhibitors of CDKs.

The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21(Cip1) at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21(Cip1) at Thr 145 prevents the complex formation of p21(Cip1) with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21(Cip1) at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21(Cip1) and attenuates the Cdk2 inhibitory activity of p21(Cip1). Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21(Cip1) phosphorylation is not caused by altered intracellular localization of p21(Cip1). As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21(Cip1), whereas the nonphosphorylatable p21(Cip1) T145A construct arrests cells in G(0) phase. These data suggest that the modulation of p21(Cip1) cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.

In the present study, we report the cyclin-dependent kinase (Cdk)-inhibitory activity of a series of p21waf1/cip1 (p21) peptide fragments spanning the whole protein against the cyclin D1/Cdk4 and cyclin E/Cdk2 enzymes. The most potent p21 peptide tested in our initial peptide series, designated W10, spanned amino acids 139 to 164, a region of p21 that has been found independently to bind to proliferating cell nuclear antigen and also to inhibit Cdk activity. We go on to report the importance of putative beta-strand and 3(10)-helix motifs in the W10 peptide for cyclin-dependent kinase-inhibitory activity. We also describe the cellular activity of W10 and derivatives that were chemically linked to an antennapedia peptide, the latter segment acting as a cell membrane carrier. We found that the W10AP peptide exhibited growth inhibition that resulted from necrosis in human lymphoma CA46 cells. Furthermore, regions in the W10 peptide responsible for Cdk-inhibition were also important for the degree of this cellular activity. These studies provide insights that may eventually, through further design, yield agents for the therapy of cancer.

Peptides derived from mutated ras are immunogenic in mice and humans, and represent a group of specific tumor antigens that are potential targets for immunotherapy. T-cell responses against mutant p21 ras can be initiated in vitro by repeated stimulation of peripheral-blood mononuclear cells with mutant ras-derived peptides. Patients with tumors commonly harbouring ras mutations may therefore show evidence of in vivo reactivity against such mutations. Peripheral-blood mononuclear cells from 10 patients with colorectal adenocarcinoma were screened for reactivity against synthetic ras-derived peptides corresponding to the most commonly found mutations in this type of cancer. In one patient, T-cell reactivity against the 1-25,13Gly-->Asp peptide was detected. From this patient, both CD4+ and CD8+ T-cell clones specific for the 1-25,13Gly-->Asp mutation could be raised. We were not, however, able to detect the corresponding mutation in the cancer. The 13Gly-->Asp mutation in the ras oncogene is frequent and constitutes 9 to 27% of all K ras mutations found in biopsies from patients with colorectal carcinomas. Our study demonstrates a mutant ras-specific T-cell response of both the CD4+ and the CD8+ phenotype in a cancer patient. We speculate that in this patient a specific T-cell response resulted in eradication of tumor cells harboring the 13Gly-->Asp mutation.