Summary Epidemiologic studies indicate that the risks for major age-related debilities including CHD, diabetes, and age-related macular degeneration (AMD) are diminished in people who consume lower glycemic index (GI) diets but lack of a unifying physiobiochemical mechanism that explains the salutary effect is a barrier to implementing dietary practices that capture the benefits of consuming lower GI diets. We established a simple murine model of age-related retinal lesions that precede AMD (hereafter called AMD-like lesions). We found that consuming a higher GI diet promotes these AMD-like lesions. However, mice that consumed the lower vs. higher GI diet had significantly reduced frequency (p<0.02) and severity (p<0.05) of hallmark age-related retinal lesions such as basal deposits. Consuming higher GI diets was associated with >3 fold higher accumulation of advanced glycation end products (AGEs) in retina, lens, liver and brain in the age-matched mice, suggesting diet-induced systemic glycative stress that is etiologic for lesions. Data from live cell and cell free systems show that the ubiquitin-proteasome system (UPS) and lysosome/autophagy pathway (LPS) are involved in the degradation of AGEs. Glycatively-modified substrates were degraded significantly slower than unmodified substrates by the UPS. Compounding the detriments of glycative stress, AGE-modification of ubiquitin and ubiquitin conjugating enzymes impaired UPS activities. Furthermore, ubiquitin conjugates and AGEs accumulate and are found in lysosomes when cells are glycatively stressed or the UPS or LPS/autophagy are inhibited indicating that the UPS and LPS interact with one another to degrade AGEs. Together these data explain why AGEs accumulate as glycative stress increases.

Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high affinity binding of ubiquitin chains, but in their absence ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2-4 fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP) to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded.

Ribonucleotide reductase catalyzes a crucial step in de novo DNA synthesis and is allosterically controlled by relative levels of dNTPs to maintain a balanced pool of deoxynucleoside triphosphates in the cell. In eukaryotes, the enzyme comprises a heterooligomer of ␣2 and ␤2 subunits. The ␣ subunit, Rnr1, contains catalytic and regulatory sites. Here, we report the only x-ray structures of the eukaryotic ␣ subunit of ribonucleotide reductase from Saccharomyces cerevisiae. The structures of the apo-, AMPPNP only-, AMPPNP-CDP-, AMPPNP-UDP-, dGTP-ADP-and TTP-GDP-bound complexes give insight into substrate and effector binding and specificity cross-talk. These are Class I structures with the only fully ordered catalytic sites, including loop 2, a stretch of polypeptide that spans specificity and catalytic sites, conferring specificity. Binding of specificity effector rearranges loop 2; in our structures, this rearrangement moves P294, a residue unique to eukaryotes, out of the catalytic site, accommodating substrate binding. Substrate binding further rearranges loop 2. Cross-talk, by which effector binding regulates substrate preference, occurs largely through R293 and Q288 of loop 2, which are analogous to residues in Thermotoga maritima that mediate cross-talk. However loop-2 conformations and residue-substrate interactions differ substantially between yeast and T. maritima. In most effector-substrate complexes, water molecules help mediate substrate-loop 2 interactions. Finally, the substrate ribose binds with its 3 hydroxyl closer than its 2 hydroxyl to C218 of the catalytic redox pair. We also see a conserved water molecule at the catalytic site in all our structures, near the ribose 2 hydroxyl.ribonucleotide reductase ͉ allosteric regulation ͉ crystallography ͉ DNA synthesis ͉ dNTP pools E ukaryotic ribonucleotide reductase (RNR) is an enzyme composed of ␣ 2 and ␤ 2 subunits that catalyzes a crucial step of de novo DNA synthesis by converting nucleoside diphosphates to deoxynucleoside diphosphates (1, 2). Tight control of dNTP pools is vital for cell viability; because of the crucial role RNR plays in balancing the relative levels of dNTPs, it is highly regulated transcriptionally (3), allosterically (4-6), by compartmentalization of the various subunits within the cell (7,8), and, in Saccharomyces cerevisiae, by its protein inhibitor Sml1 (9-12). The molecular basis for these processes is not fully understood.Rnr1, the ␣ subunit of RNR, contains the catalytic site, the substrate-specificity site, and the activity site (Fig. 1A), whereas the ␤ subunit houses a tyrosyl radical required for RNR activity (13,14). An elegant mechanism of specificity cross-talk determines substrate preference based on the nucleotide effector bound at the specificity site (4, 15, 16). Brown and Reichard (2) have proposed that the effectors ATP and dATP bind at the activity site and activate or inhibit, respectively. They also bind at the specificity site and select for pyrimidine substrates, whereas thymin triphosphate (TTP) and dGT...

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. Crucial for rapidly dividing cells, RNR is a target for cancer therapy. In eukaryotes, RNR comprises a heterooligomer of ␣2 and ␤2 subunits. Rnr1, the ␣ subunit, contains regulatory and catalytic sites; Rnr2, the ␤ subunit (in yeast, a heterodimer of Rnr2 and Rnr4), houses the diferric-tyrosyl radical crucial for catalysis. Here, we present three x-ray structures of eukaryotic Rnr1 from Saccharomyces cerevisiae: one bound to gemcitabine diphosphate (GemdP), the active metabolite of the mechanism-based chemotherapeutic agent gemcitabine; one with an Rnr2-derived peptide, and one with an Rnr4-derived peptide. Our structures reveal that GemdP binds differently from its analogue, cytidine diphosphate; because of unusual interactions of the geminal fluorines, the ribose and base of GemdP shift substantially, and loop 2, which mediates substrate specificity, adopts different conformations when binding to GemdP and cytidine diphosphate. The Rnr2 and Rnr4 peptides, which block RNR assembly, bind differently from each other but have unique modes of binding not seen in prokaryotic RNR. The Rnr2 peptide adopts a conformation similar to that previously reported from an NMR study for a mouse Rnr2-based peptide. In yeast, the Rnr2 peptide binds at subsites consisting of residues that are highly conserved among yeast, mouse, and human Rnr1s, suggesting that the mode of Rnr1-Rnr2 binding is conserved among eukaryotes. These structures provide new insights into subunit assembly and a framework for structure-based drug design targeting RNR.allosteric regulation ͉ crystallography ͉ dNTP ͉ chemotherapy ͉ gemcitabine R ibonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, essential precursors of DNA synthesis. Crucial for rapidly proliferating cells, RNR is a target for anticancer (1, 2) and antiviral (2, 3) drugs. Gemcitabine, an analogue of deoxycytidine (2Ј-2Ј-difluorodeoxycytidine), is sequentially phosphorylated to the 5Ј-monophosphate form by deoxycytidine kinase and to difluorodeoxycytidine 5Ј-diphosphate (GemdP) by uridylate-cytidylate monophosphate kinase. In the presence of reductants, GemdP inactivates Rnr1. In the absence of reductants, with prereduced Rnr1 and Rnr2, inhibition occurs from the loss of the tyrosyl radical in Rnr2 (1). Recently, GemdP has been shown to inactivate both human R1 and R2 (JoAnne Stubbe, personal communication). Inhibition of RNR by GemdP leads to reduction of the pool of deoxyribonucleotide 5Ј-diphosphates available for DNA synthesis, presumably favoring incorporation of the gemcitabine triphosphate metabolite by DNA polymerase ␣, preventing chain elongation (4, 5).RNRs require unusual metallocofactors to initiate radicalbased nucleotide reduction and are divided into three classes based on their cofactor. Class I RNR, found in all eukaryotes, is a heterooligomer of ␣ 2 and ␤ 2 subunits (6). In eukaryotes, the ␣ subunit, called Rnr1, c...

Ubiquitin (Ub)-protein conjugates formed by purified ringfinger or U-box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST-fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST-UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non-forked Ub conjugates.