Human mitochondrial pyrroline-5-carboxylate reductase (PYCR) is a house-keeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate to proline. This enzymatic cycle plays pivotal roles in amino acid metabolism, intracellular redox potential and mitochondrial integrity. Here, we hypothesize that PYCR1 might be a novel prognostic biomarker and therapeutic target for breast cancer. In this study, breast cancer tissue samples were obtained from Zhejiang University (ZJU set). Immunohistochemistry analysis was performed to detect the protein level of PYCR1, and Kaplan-Meier and Cox proportional analyses were employed in this outcome study. The prognostic significance and performance of PYCR1 mRNA were validated on 13 worldwide independent microarray data sets, composed of 2500 assessable breast cancer cases. Our findings revealed that both PYCR1 mRNA and protein expression were significantly associated with tumor size, grade and invasive molecular subtypes of breast cancers. Independent and pooled analyses verified that higher PYCR1 mRNA levels were significantly associated with poor survival of breast cancer patients, regardless of estrogen receptor (ER) status. For in vitro studies, inhibition of PYCR1 by small-hairpin RNA significantly reduced the growth and invasion capabilities of the cells, while enhancing the cytotoxicity of doxorubicin in breast cancer cell lines MCF-7 (ER positive) and MDA-MB-231 (ER negative). Further population study also validated that chemotherapy significantly improved survival in early-stage breast cancer patients with low PYCR1 expression levels. Therefore, PYCR1 might serve as a prognostic biomaker for either ER-positive or ER-negative breast cancer subtypes and can also be a potential target for breast cancer therapy.

A novel RNA recognition motif is characterized in an arginine-rich peptide. The motif, derived from lambda transcriptional antitermination protein N, regulates an RNA-directed genetic switch. Its characterization by multidimensional nuclear magnetic resonance (NMR) demonstrates specific RNA-dependent folding of N- and C-terminal recognition helices separated by a central bend. The biological importance of the bent alpha-helix is demonstrated by mutagenesis: binding is blocked by substitutions in the N peptide or its target (the boxB RNA hairpin) associated in vivo with loss of transcriptional antitermination activity. Although arginine side chains are essential, the peptide is also anchored to boxB by specific nonpolar contacts. An alanine in the N-terminal helix docks in the major groove of the RNA stem whereas a tryptophan in the C-terminal helix stacks against a purine in the RNA loop. At these positions all 19 possible amino acid substitutions have been constructed by peptide synthesis; each impairs binding to boxB. The pattern of allowed and disallowed substitutions is in accord with the results of random-cassette mutagenesis in vivo. The helix-bend-helix motif rationalizes genetic analysis of N-dependent transcriptional antitermination and extends the structural repertoire of arginine-rich domains observed among mammalian immunodeficiency viruses.

Sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent protein lysine modifying enzymes with deacetylase, adenosine diphosphateribosyltransferase and other deacylase activities. Mammals have seven sirtuins, namely SIRT1-7. They are key regulators for a wide variety of cellular and physiological processes such as cell proliferation, differentiation, DNA damage and stress response, genome stability, cell survival, metabolism, energy homeostasis, organ development, aging, and cancer. Here we present an extensive literature review of the roles of mammalian sirtuins, particularly SIRT1 as that is the most studied sirtuin, in human epithelial, neuronal, hematopoietic, and mesenchymal malignancies, covering breast, prostate, lung, thyroid, liver, colon, gastric, pancreatic, ovarian, and cervical cancers, tumors of the central nervous system, leukemia and lymphoma, and soft tissue sarcomas. Collective evidence suggests sirtuins are involved in both promoting and suppressing tumorigenesis depending on cellular and molecular contexts. We discuss the potential use of sirtuin modulators, especially sirtuin inhibitors, in cancer treatment.

Ribonucleotide reductase (RNR) is an attractive target for anticancer agents given its central function in DNA synthesis, growth, metastasis, and drug resistance of cancer cells. The current clinically established RNR inhibitors have the shortcomings of short halflife, drug resistance, and iron chelation. Here we report the development of a novel class of effective RNR inhibitors addressing these issues. A novel ligand-binding pocket on the RNR small subunit (RRM2) near the C-terminal tail was proposed by computer modeling and verified by site-directed mutagenesis and NMR techniques. A compound targeting this pocket was identified by virtual screening of the NCI diverse small molecule database. By lead optimization we developed the novel RNR inhibitor COH29 which acted as a potent inhibitor of both recombinant and cellular human RNR enzymes. COH29 overcame hydroxyurea and gemcitabine resistance in cancer cells. It effectively inhibited proliferation of most cell lines in the NCI 60 human cancer panel, most notably ovarian cancer and leukemia, but exerted little effect on normal fibroblasts or endothelial cells. In mouse xenograft models of human cancer, COH29 treatment reduced tumor growth compared to vehicle. Site-directed mutagenesis, NMR and surface plasmon resonance biosensor studies confirmed COH29 binding to the proposed ligand-binding pocket and offered evidence for assembly blockade of the RRM1-RRM2 quaternary structure. Our findings offer preclinical validation of COH29 as a promising new class of RNR inhibitors with a new mechanism of inhibition, with broad potential for improved treatment of human cancer.

Ribonucleotide reductase small subunit B (RRM2B) is a stress response protein that protects normal human fibroblasts from oxidative stress. However, the underlying mechanism that governs this function is not entirely understood. To identify factors that interact with RRM2B and mediate anti-oxidation function, large-scale purification of human Flag-tagged RRM2B complexes was performed. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1, PYCR2) were identified by mass spectrometry analysis as components of RRM2B complexes. Silencing of both PYCR1 and PYCR2 by expressing short hairpin RNAs induced defects in cell proliferation, partial fragmentation of the mitochondrial network, and hypersensitivity to oxidative stress in hTERT-immortalized human foreskin fibroblasts (HFF-hTERT). Moderate overexpression of RRM2B, comparable to stress-induced level, protected cells from oxidative stress. Silencing of both PYCR1 and PYCR2 completely abolished anti-oxidation activity of RRM2B, demonstrating a functional collaboration of these metabolic enzymes in response to oxidative stress.