P16INK4A (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly due to the prevalence of genetic inactivation of the p16INK4A (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that elevated expression (up-regulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications in the structure-function relationship of P16 and in human cancers.

Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 25% of clinically used drugs. Genetic polymorphisms cause substantial variation in CYP2D6 activity and serve as biomarkers guiding drug therapy. However, genotype-phenotype relationships remain ambiguous except for poor metabolizers carrying null alleles, suggesting the presence of yet unknown genetic variants. Searching for regulatory CYP2D6 polymorphisms, we find that a SNP defining the CYP2D6*2 allele, rs16947 [R296C, 17-60% minor allele frequency (MAF)], previously thought to convey normal activity, alters exon 6 splicing, thereby reducing CYP2D6 expression at least 2-fold. In addition, two completely linked SNPs (rs5758550/rs133333, MAF 13-42%) increase CYP2D6 transcription more than 2-fold, located in a distant downstream enhancer region (>100 kb) that interacts with the CYP2D6 promoter. In high linkage disequilibrium (LD) with each other, rs16947 and the enhancer SNPs form haplotypes that affect CYP2D6 enzyme activity in vivo. In a pediatric cohort of 164 individuals, rs16947 alone (minor haplotype frequency 28%) was associated with reduced CYP2D6 metabolic activity (measured as dextromethorphan/metabolite ratios), whereas rs5758550/rs133333 alone (frequency 3%) resulted in increased CYP2D6 activity, while haplotypes containing both rs16947 and rs5758550/rs133333 were similar to the wild-type. Other alleles used in biomarker panels carrying these variants such as CYP2D6*41 require re-evaluation of independent effects on CYP2D6 activity. The occurrence of two regulatory variants of high frequency and in high LD, residing on a long haplotype, highlights the importance of gene architecture, likely shaped by evolutionary selection pressures, in determining activity of encoded proteins.

Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclindependent kinase 4) family have been reported recently, the detailed structures and the functional roles of the loops have drawn considerable interest. This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects. First, the solution structure of p18 INK4C was determined by NMR, and the loop structures were analyzed in detail. The loops adapt nascent antiparallel -sheet structures, but the positions are slightly different from those in the crystal structure. A detailed comparison between the solution structures of p16 and p18 has also been presented. The determination of the p18 solution structure made such detailed comparisons possible for the first time. Second, the { 1 H, 15 N}HSQC NMR experiment was used to probe the interactions between p18 INK4C and other proteins. The results suggest that p18 INK4C interacts very weakly with dna K and glutathione S-transferase via the loops. The third aspect employed site-specific mutagenesis and functional assays. Three mutants of p18 and 11 mutants of p16 were constructed to test functional importance of loops and helices. The results suggest that loop 2 is likely to be part of the recognition surface of p18 INK4C or p16 INK4A for CDK4, and they provide quantitative functional contributions of specific residues. Overall, our results enhance understanding of the structural and functional roles of the loops in INK4 tumor suppressors in particular and in ankyrinrepeat proteins in general.The structure and function of the INK4 family of tumor suppressors are active subjects of current research in many laboratories. There are four known members in this family: p15 INK4B

Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and for the K53,56,120M triple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the interface as well as the substrate binding to the enzyme at the interface. The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2.

Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C′, and C R ) and all heavy atoms (residues 4-224) of 0.69 ( 0.09 and 1.04 ( 0.09 Å, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16 INK4A , possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16 INK4A identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N-and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.Human gankyrin is a liver oncoprotein that plays an important role in the development of hepatocellular carcinomas (1). Overexpression of gankyrin has been observed in all studied human and rodent hepatocellular carcinomas, and has been further correlated with increases in the levels of both phosphorylation and degradation of tumor suppressor retinoblastoma protein (Rb) (1-3). In relation to these effects, gankyrin has been shown to interact with three components in the Rb pathway, namely, Rb, the S6 ATPase subunit of the 26S proteasome, and cyclin-dependent kinase 4 (CDK4) 1 (1,4,5). From in Vitro studies, dual functional roles have been proposed for gankyrin in the expanded INK4-CDK4/6-Rb pathway (5). On one hand, gankyrin binds to both tumor suppressor Rb and the S6 ATPase subunit of the 26S proteasome, which in turn facilitates CDK4/6-mediated Rb phosphorylation and drives Rb into ubiquitin-mediated degradation. On the other hand, gankyrin directly interacts with CDK4 and counteracts the inhibitory function of p16 INK4A and p18 INK4C. Moreover, it has been shown that the oncogenic activity of gankyrin can be suppressed by binding to melanoma antigen (MAGE)-A4, a tumor specific antigen with potential in antitumor immunotherapy (6). These findings taken together strongly suggest that gankyrin is a promising target for developing novel therapeutic strategies a...

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Purpose As the result of a recent national shortage in paclitaxel, some patients who were receiving or scheduled to receive weekly paclitaxel were converted to every 3-week (q3w) docetaxel with granulocyte colony-stimulating factor support. Our institution noted higher than expected incidence of severe skin toxicity events attributable to docetaxel during the shortage period among our breast cancer patients. In this report, we summarize the clinical course of the first five cases, review the literature surrounding docetaxel-induced skin toxicity, and offer possible prevention and treatment strategies to improve docetaxel tolerability. Methods The observation period for this case series was August 1 through October 21, 2011. All patients treated with docetaxel were identified from our electronic medical record. Operable stage I–III breast cancer patients who received ≥1 dose of docetaxel monotherapy at 75–100 mg/m2 q3w were included in this study. The cases of grade 3–4 docetaxel-induced skin toxicities identified by the treating oncologists were then contacted and signed an informed consent through an Institutional Review Board-approved protocol. Results Thirty-four patients met the inclusion criteria. Five patients (14.7 %) experienced grade 3 skin toxicity events attributable to docetaxel, a significantly higher rate than previously reported for docetaxel dosed at 75–100 mg/m2. Conclusions Docetaxel-induced dermatologic toxicity is well characterized; nonetheless, its etiology is largely unknown and evidence-based prevention and management strategies are lacking. This report shows that the use of docetaxel 75–100 mg/m2 q3w subsequent to dose-dense doxorubicin and cyclophosphamide regimen can lead to unacceptable rate of severe skin toxicity.

We report the first detailed structure-function analyses of p18 INK4C (p18), which is a homologue of the important tumor suppressor p16 INK4A (p16). Twenty-four mutants were designed rationally. The global conformations of the mutants were characterized by NMR, while the function was assayed by inhibition of cyclin-dependent kinase 4 (CDK4). Most of these mutants have unperturbed global structures, thus the changes in their inhibitory abilities can be attributed to the mutated residues. The important results are summarized as follows: (a) some residues at loops 1 and 2, but not 3, are important for the inhibitory function of p18, similar to the results for p16; (b) two residues at the first helix-turn-helix motif and two at the third are important for inhibition; (c) while the results generally agree with the prediction based on the crystal structures of p16-CDK6 and p19-CDK6 binary complexes, there are significant differences in a few residues, suggesting that the interactions in the binary complexes may not accurately represent the interactions in the ternary complexes (in the presence of cyclin D2); (d) most importantly, the extra loop of p18 appears to contribute to the function of p18, even though the crystal structure of the p19 INK4D -CDK6 complex indicates no interactions involving this loop; (e) detailed analyses of the crystal structures and the functional results suggest that there are notable differences in the interactions between different members of the INK4 family and CDKs. INK41 proteins are a group of ankyrin repeat-containing proteins, which specifically bind to and inactivate cyclindependent kinases 4 and 6 (1-6). Currently, there are four known members of the INK4 family, p16INK4A , p15 INK4B , p18 INK4C , and p19 INK4D (which are abbreviated as p16, p15, p18, and p19, respectively, in this paper) (7-13). While p15 and p16 consist of four ankyrin repeats (ankyrin I-IV), p18 and p19 have five (ankyrin I-V). An ankyrin repeat is a motif of about 34 amino acid residues and is present in a large number of proteins. Ankyrin repeats exist in groups of four or more and are often involved in protein-protein interaction (14,15). Since 1997, the tertiary structures of p19 (16), p18 (17, 18), p16 (19), and p15 (20) (15,16,19,20). Figure 1 shows the correlation between p16, p15, p18, and p19 in terms of residue numbers and the locations of helices (e.g., helix IA stands for the first helix of the first ankyrin) and loops (e.g., loop 1 stands for the first loop, connecting ankyrin I and ankyrin II). Another major advance in the field is the recent solution of the crystal structures of p16-CDK6 (21) and p19-CDK6 (22) binary complexes. In the crystal structure of the p16-CDK6 complex, p16 binds to one side of the catalytic cleft of CDK6, opposite the cyclin-binding site. The concave surface of p16 contacts the N lobe of CDK6, while the loops between neighboring ankyrin repeats interact with the C lobe. As a result, the interactions between p16 and CDK6 involve multiple regions in both proteins. M...