<p>Gene duplication is an important evolutionary tool for fostering diversification and expanding gene families. However, while this concept is well understood and accepted in a theoretical capacity, the particular changes that lead to the functional diversification of gene duplicates are less well understood and documented. Additionally, little work has been done to understand how functions are gained or lost, which leads to the diversification of orthologous genes. The Sir2 family of NAD+-dependent deacetylases is an excellent gene family to study questions of duplication and diversification as it is ubiquitous throughout all kingdoms of life, and it has expanded through a number of gene duplications so that while most bacteria have a single sirtuin/species, mammals have seven sirtuins/species. Sirtuins also have a wide array of biological functions and targets, but some of these functions are conserved in eukaryotes.</p><p>In this study, Sir2 is used to investigate the principles behind gene duplication and functional diversification in a molecular context. Sir2 function is studied in multiple species of budding yeast, the model organism Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida lusitaniae using a combination of genetic, biochemical, and high-throughput methods. Sir2 and its paralog Hst1 from S. cerevisiae were used with their non-duplicated ortholog Sir2 from K. lactis to examine the type of molecular changes that occur after gene duplication and lead to subfunctionalization. Then Sir2 from the more divergent C. lusitaniae was used to study how functions are gained or lost.</p><p>To study the molecular mechanism of subfunctionalization in the duplicated deacetylases ScSir2 and ScHst1 with the non-duplicated KlSir2 used as a proxy for the ancestral state, we hypothesized that the basis for subfunctionalization in this case was in the interaction domains. ScSir2 and ScHst1 act in distinct complexes that target them to the genomic loci they regulate. KlSir2 interacts with the same complexes as both ScSir2 and ScHst1. Therefore, we first identified the minimal regions of ScSir2 and ScHst1 necessary for each to interact with its respective complex. Then we identified mutations in those interaction domains that eliminated those interactions. Those mutations were then tested in KlSir2 for their impact on its interactions with the same complexes. We found that the interaction domains in ScSir2 and ScHst1 were conserved in KlSir2, demonstrating that Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.</p><p>To understand better how Sir2 has gained or lost functions, we studied the Sir2 function in C. lusitaniae to serve as an intermediate between the fission yeast Schizosaccharomyces pombe Sir2, whose functions have been identified, and K. lactis and S. cerevisiae. Interestingly, ClSir2 was localized to the rDNA, which is also the case in S. pombe, K. lactis, and S. cerevisiae, but not at the telomeres, which is another locus at which Sir2 is found in other yeast. Additionally, ClSir2 was not found to have an impact on gene expression unlike Sir2 and Hst1 in other yeast where they repress transcription.</p> / Dissertation

Silent Information Regulator 2 (SIR2) have a phylogenetically conserved catalytic domain from bacteria to humans. It catalyzes NAD+ dependent deacetylase activity post-translationally on acetylated lysine residues present in the protein. Because SIR2 are NAD+ dependent, its activity gets influenced by the change in the level of NAD+. SIR2 is responsible for calorie restriction and increased replicative yeast lifespan. It breakdown high energy bond in nicotinamide adenine dinucleotide (NAD), and the synthesis of O-acetyl-ADP-ribose which is a novel product. Lysine de/acetylation of histone molecule plays a significant role in chromatin dynamics in eukaryotes, but little is known in term of non-histone molecule modification by SIR2 enzyme especially in the case of the plant. SIP-428 is one of the SABP2 interacting protein (SIP) that exhibit SIR2 deacetylase activity. SABP2 is one of the essential components of salicylic acid (SA) signaling pathway that converts inactive methyl salicylate (MeSA) to active SA to induce local as well as SAR. AtSRT2, an Arabidopsis homolog of SIP-428 negatively regulate the basal resistance. Although catalytic domain is conserved, functional divergence has been reported in the case of SIR2 homologs. Presence of acetylated lysine residue in many cellular and organellar proteins implicated the possible physiological and metabolic role of SIP-428. Our result demonstrated SIP-428 exhibited NAD+ dependent deacetylase activity, but its lysine residue found to be acetylated, which raises the possibility of a post-translation regulatory mechanism that modulates the activity of SIP-428. SIP-428 have non-histone substrate, the negative regulator of basal resistance, and SAR. To understand better about the role of SIP-428 in plant physiology how it plays a vital role in SABP2 signaling pathway we will be using transgenic tobacco plant with altered expression of SIP-428 (Silence and inducible overexpression). Verified T3 generation of silence line and T2 generation of overexpression were created. These transgenic plant will be used to answer the possible link between SIP-428 and SABP2 in response to pathogen infection.

Abiotic stress leads to a change in the water content of plants. Salinity and osmotic stress affect both the morphology and physiology of plants. Plants have therefore responded to these environmental changes by adapting and tolerating them. The SABP2-interacting proteins (SIP) 428-silenced RNAi transgenic tobacco lines were subjected to various abiotic stresses (salinity, osmotic, and drought). The effect of SIP428-silencing on the tobacco plants subjected to these abiotic stresses was monitored. The results from the root growth data show that the sip428-silenced lines exhibit enhanced tolerance to the stressors compared to the wild-type plants. Interestingly, results of the relative chlorophyll content show no significant difference between the wild-type plants and sip428-silenced transgenic plants. In summary, based on the results presented in this study it could be concluded that SIP428 is a negative regulator of salinity, osmotic and drought stresses. Further studies are required to understand the mechanism.

Abiotic stresses are constantly rising and pose a very high risk to global agricultural productivity and food security. Some plants have evolved several innate pathways for defense against these stresses. Hence, understanding stress signaling pathways can help develop crop plants with higher stress tolerance. The salicylic acid-mediated signaling pathway is important in plants experiencing biotic and abiotic stresses. In previous studies, SABP2-Interacting Protein (SIP-428) has been shown to be a negative regular of plant growth under abiotic stress. This study aimed to investigate the roles of SIP-428 in the ROS signaling of tobacco plants. We investigated transgenic RNAi-silenced lines of SIP-428 and wild-type tobacco plants for the activities of guaiacol peroxidase and catalase enzymes in Mannitol and NaCl-stressed plants for 7 and 14 days. Our results showed that SIP-428 plays a significant role in ROS signaling in Mannitol and NaCl-stressed plants via the activities of guaiacol peroxidase.

Yeasts are unicellular eukaryotic microorganisms, capable of forming of organised multicellular communities, the colonies. Many yeast strains possess a characteristic colony morphology under defined living conditions. Another feature typical for many feral and pathogenic yeast strains is the ability to switch their morphotype. This phenomenon, called the phenotypic switching, contributes to a rapid adaptation to the changing harmful environment and is often connected with changes of the stress resistance or with the changes of virulence of pathogenic yeasts. Phenotypic switching can be observed even in non-pathogenic yeast Saccharomyces cerevisiae. The strain BR-F, isolated from nature, switches under laboratory conditions from fluffy to smooth morphology of the strain BR-S. This phenotypic switch is accompanied by broad changes in the phenotype. Transcriptome analyses of the strains BR-F and BR-S have shown, among others, changes in expression of the subtelomeric genes that are under control of the histone acetylases and deacetylases. My work was aimed to the histone deacetylase Sir2p, which could influence the phenotypic switching in Saccharomyces cerevisiae. The sir2 deletion mutant of the strain BR-S, prepared in our laboratory, was used for my studies. The results show, that the strain BR-S...

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant known to accumulate less SA. Taken together expression of SBIP-428 is negatively correlated to the levels of SA in plants. The AtSRT2 plants exhibit no altered growth phenotype but exhibit a higher pathogen resistance against bacterial pathogen. Our results indicate that SBIP-428 is an important regulator in plant defense pathway.

The NAD-dependent deacetylase SIRT1 regulates several factors involved in stress response and cell survival but its function in cancer is largely unknown. Research suggests that SIRT1 influences several transcription factors and molecules that are important components of pathways often deregulated in cancer. Our experiments have shown that SIRT1 knock down by short hairpin RNA accelerates tumor xenograft formation by HCT116 colon cancer cells, whereas SIRT1 overexpression inhibits tumor formation. We have also found that, pharmacological inhibition of SIRT1 stimulates cell proliferation under conditions of growth factor deprivation suggesting a tumor suppressive function of SIRT1. Paradoxically, SIRT1 inhibition sensitizes the same cells to apoptosis by chemotherapeutic drugs. Immunohistochemical staining of a colon tumor microarray revealed high SIRT1 expression levels in normal colon mucosa and benign adenomas. SIRT1 overexpression was observed in nearly 25% of stage I/II/III colorectal adenocarcinomas but rarely found in advanced stage IV tumors. Furthermore, about 30% of carcinomas showed lower than normal SIRT1 expression. These results suggest a pleiotropic effect of SIRT1 in cancer, i.e., anti-proliferative as well as anti-apoptotic. Further experiments along these lines and examination of a larger patient cohort could provide a rationale for the use of SIRT1 activators and inhibitors in the prevention and treatment of cancer.

Salicylic acid is widely studied for its role in biotic stress signaling in plants. Several SA-binding proteins, including SABP2 (salicylic acid-binding protein 2) has been identified and characterized for their role in plant disease resistance. SABP2 is a 29 kDA tobacco protein that binds to salicylic acid with high affinity. It is a methylesterase enzyme that catalyzes the conversion of methyl salicylate into salicylic acid required for inducing a robust systemic acquired resistance (SAR) in plants. Methyl salicylic acid is one of the several mobile SAR signals identified in plants. SABP2-interacting protein 428 (SIP428) was identified in a yeast two-hybrid screen using tobacco SABP2 as a bait. In silico analysis shows that SIP428 possesses the SIR2 (silent information regulatory 2)-like conserved motifs. SIR2 enzymes are orthologs of sirtuin proteins that catalyze the NAD+-dependent deacetylation of Nε lysine-acetylated proteins. The recombinant SIP428 expressed in E. coli exhibits SIR2-like deacetylase activity. SIP428 shows homology to Arabidopsis AtSRT2 (67% identity), which is implicated in SA-mediated basal defenses. Immunoblot analysis using anti-acetylated lysine antibodies showed that the recombinant SIP428 is lysine acetylated. The expression of SIP428 transcripts was moderately downregulated upon infection by TMV. In the presence of SIP428, the esterase activity of SABP2 increased modestly. The interaction of SIP428 with SABP2, it's regulation upon pathogen infection, and similarity with AtSRT2 suggests that SIP428 is likely to play a role in stress signaling in plants.

In Saccharomyces cerevisiae, telomeric and HM silencing requires the histone deacetylase Sir2 and the chromatin binding proteins Sir3 and Sir4, which interact to form the SIR complex. Silent chromatin formation begins with a nucleation step, followed by spreading of Sir proteins along chromatin. Overexpression of Sir3 extends silent chromatin domains, however the role of Sir protein interactions within silent chromatin extensions remains unknown. Here, we generated the Sir3 mutant, Sir3-4A, which cannot interact with Sir4 but is capable of forming silent chromatin extensions when overexpressed. Within extended silent domains, Sir2 and Sir4 enrichments are similar whether Sir3 or Sir3-4A is overexpressed, suggesting that silent chromatin extensions require Sir4 but not the interaction between Sir3 and Sir4. Tethering Sir3-4A at an HMR silencer cannot nucleate silencing in the absence of Sir3, suggesting that in addition to Sir3 recruitment, the Sir3-Sir4 interaction has at least one other function during silent chromatin nucleation.

SABP2 (Salicylic Acid Binding Protein 2) plays a vital role in the salicylic acid signaling pathway of plants both regarding basal resistance and systemic acquired resistance against pathogen infection. SIP-428 (SABP2 Interacting Protein-428) is a Silent information regulator 2 (SIR2) like deacetylase enzyme that physically interacts with SABP2 in a yeast two-hybrid interaction and confirmed independently by a GST pull-down assay. We demonstrated that SIP- 428 is an NAD+ dependent SIR2 deacetylase enzyme. Transgenic tobacco plants silenced in SIP- 428 expression via RNAi showed enhanced basal resistance to microbial pathogens. Moreover, these SIP-428-silenced lines also exhibited a robust induction of systemic acquired resistance. In contrast, the transgenic tobacco lines overexpressing SIP-428 showed compromised basal resistance and failed to induce systemic acquired resistance. These results indicate that SIP-428 is likely a negative regulator of SA-mediated plant immunity. Experiments using a SABP2 inhibitor showed that SIP-428 likely functions upstream of SABP2 in the salicylic acid signaling pathway. It also indicates that SABP2 is dependent on SIP-428 for its role in the SA signaling pathway. Subcellular localization studies using confocal microscopy and subcellular fractionation showed that SIP-428 localized in the mitochondria. These results clearly show a role for SIP-428 in plant immunity.