Structure-Function and Regulation of Yeast Ribonucleotide Reductase Inhibitor, Sml1

Sml1 is a small protein in Saccharomyces cerevisiae that inhibits the activity of ribonucleotide reductase (RNR) through its interactions with the large subunit of RNR. RNR catalyzes the reduction of nucleotide diphosphates (NDPs) to deoxynucleotide diphosphates (dNDPs) that is the rate-limiting step of de novo deoxynucleotide triphosphate (dNTP) synthesis. The cellular level of Sml1 is regulated by DNA damage and replication block response through its phosphorylation by the Dun1 kinase. The goal of this dissertation research is to elucidate structure-function and regulation of Sml1. First, biochemical characterization of recombinant Sml1 was conducted using mass spectrometry and gel filtration chromatography (Chapter 3 and 4). The data shows that a disulfide bond and non-covalent interactions mediate Sml1 oligomerization. Furthermore, alkali metal adducts (Na+/K+) that bind strongly with Sml1 were found. Second, the phosphorylation of Sml1 by the Dun1 kinase was studied by a combination of mass spectrometry, site directed mutagenesis, and P32 incorporation (Chapter 5). Three phosphorylation sites of Sml1 (Ser56, Ser58 and Ser60) were identified. The data also reveals that the Dun1 kinase requires an acidic residue at the +3 position and there is cooperativity between the phosphorylation sites. Third, the relationship between Sml1 phosphorylation and the Sml1-Rnr1 interactions were investigated based on P32 incorporation, fluorescence spectroscopy, and a RNR activity assay (Chapter 6). We demonstrated that the Sml1-Rnr1 interactions reduced the phosphorylation levels of Sml1 by making the phosphorylation sites less accessible. Our data also suggest that phosphorylation of Sml1 weakens the ability of Sml1 to inhibit RNR. Taken together, this work has provided in-depth insights of Sml1’s structure-function and regulation

Nihon University CubeSat Program

The CubeSat program is now proceeding in Japan and U.S. academic institutions and radio groups. CubeSat is a 10cm cubed, 1kg weighted micro-satellite. The first launch of CubeSats is scheduled in May 2002. The second launch will be realized in autumn 2002 or later. Subsequent launch is also planned. Nihon University is going to join the second launch. Our program consists of two phases. At the first phase, we are developing a CubeSat for the second launch opportunity. The purpose of the first phase is that the students learn the whole process of the micro satellite development and operation. At the second phase, we intend to challenge some engineering mission. We have been studying on a mission of deploying an inflatable structure model. In this paper we show the latest status of the first phase of our program , and the plan for the second phase

Spd2 assists Spd1 in modulation of RNR architecture but does not regulate deoxynucleotide pools

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The S. pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with homology to Spd1. We show that Spd2 is a CRL4Cdt2 controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3ATR checkpoint dependency of CRL4Cdt2 mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4Cdt2 mutant cells to the same extent as spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, while it together with Spd2 modulates RNR architecture and sensitizes cells to DNA damage

DNA damage signalling prevents deleterious telomere addition at DNA breaks

The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage, to increase the accuracy of repair. Here we report that telomerase action is regulated as a part of the cellular response to a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity

The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response to DNA damage

Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4

A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

While ESCRT-0 is ubiquitinated by the Rsp5 E3 ligase, loss of Rsp5 does not disrupt monoubiquitin-dependent sorting into multivesicular bodies

Review of nutrient actions on age-related macular degeneration

The actions of nutrients and related compounds on age-related macular degeneration (AMD) are explained in this review. The findings from 80 studies published since 2003 on the association between diet and supplements in AMD were reviewed. Antioxidants and other nutrients with an effect on AMD susceptibility include carotenoids (lutein and zeaxanthin, β-carotene), vitamins (vitamin A, E, C, D, B), mineral supplements (zinc, copper, selenium), dietary fatty acids [monounsaturated fatty acids, polyunsaturated fatty acids (PUFA both omega-3 PUFA and omega-6 PUFA), saturated fatty acids and cholesterol], and dietary carbohydrates. The literature revealed that many of these antioxidants and nutrients exert a protective role by functioning synergistically. Specifically, the use of dietary supplements with targeted actions can provide minimal benefits on the onset or progression of AMD; however, this does not appear to be particularly beneficial in healthy people. Furthermore, some supplements or nutrients have demonstrated discordant effects on AMD in some studies. Since intake of dietary supplements, as well as exposure to damaging environmental factors, is largely dependent on population habits (including dietary practices) and geographical localization, an overall healthy diet appears to be the best strategy in reducing the risk of developing AMD. As of now, the precise mechanism of action of certain nutrients in AMD prevention remains unclear. Thus, future studies are required to examine the effects that nutrients have on AMD and to determine which factors are most strongly correlated with reducing the risk of AMD or preventing its progression

Carnosine:can understanding its actions on energy metabolism and protein homeostasis inform its therapeutic potential?

The dipeptide carnosine (β-alanyl-L-histidine) has contrasting but beneficial effects on cellular activity. It delays cellular senescence and rejuvenates cultured senescent mammalian cells. However, it also inhibits the growth of cultured tumour cells. Based on studies in several organisms, we speculate that carnosine exerts these apparently opposing actions by affecting energy metabolism and/or protein homeostasis (proteostasis). Specific effects on energy metabolism include the dipeptide's influence on cellular ATP concentrations. Carnosine's ability to reduce the formation of altered proteins (typically adducts of methylglyoxal) and enhance proteolysis of aberrant polypeptides is indicative of its influence on proteostasis. Furthermore these dual actions might provide a rationale for the use of carnosine in the treatment or prevention of diverse age-related conditions where energy metabolism or proteostasis are compromised. These include cancer, Alzheimer's disease, Parkinson's disease and the complications of type-2 diabetes (nephropathy, cataracts, stroke and pain), which might all benefit from knowledge of carnosine's mode of action on human cells. © 2013 Hipkiss et al.; licensee Chemistry Central Ltd