Prasad Tongaonkar

SIR2 Regulates Recombination between Different rDNA Repeats, but Not Recombination within Individual rRNA Genes in Yeast

It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

The DNA repair protein Rad23 is a negative regulator of multi-ubiquitin chain assembly

Rad23 is a nucleotide-excision repair protein with a previously unknown biochemical function. We determined that yeast and human Rad23 inhibited multi-ubiquitin (Ub) chain formation and the degradation of proteolytic substrates. Significantly, Rad23 could be co-precipitated with a substrate that contained a short multi-Ub chain. The UV sensitivity of rad23Δ was reduced in mutants lacking the E2 enzyme Ubc4, or the multi-Ub chain-promoting factor Ufd2. These studies suggest that the stability of proteolytic substrates is governed by the competing action of multi-Ub chain-promoting and chain-inhibiting factors. The stabilization of DNA repair and stress factors could represent an important biological function of Rad23.

Evidence for an Interaction between Ubiquitin-Conjugating Enzymes and the 26S Proteasome

ABSTRACT The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors. The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome. However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood. We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome. Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance. A catalytically inactive derivative of Ubc4 (Ubc4C86A), which causes toxicity in yeast cells, can also bind the proteasome. Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome.

Evidence for separable functions of Srp1p, the yeast homolog of importin alpha (Karyopherin alpha): role for Srp1p and Sts1p in protein degradation.

ABSTRACT Srp1p (importin α) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. Thesrp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of thesrp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressingsrp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-β-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not insrp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 insrp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.

Rhesus Macaque Theta Defensins Suppress Inflammatory Cytokines and Enhance Survival in Mouse Models of Bacteremic Sepsis

Theta-defensins (θ-defensins) are macrocyclic antimicrobial peptides expressed in leukocytes of Old World monkeys. The peptides are broad spectrum microbicides in vitro and numerous θ-defensin isoforms have been identified in granulocytes of rhesus macaques and Olive baboons. Several mammalian α- and β-defensins, genetically related to θ-defensins, have proinflammatory and immune-activating properties that bridge innate and acquired immunity. In the current study we analyzed the immunoregulatory properties of rhesus θ-defensins 1–5 (RTDs 1–5). RTD-1, the most abundant θ-defensin in macaques, reduced the levels of TNF, IL-1α, IL-1β, IL-6, and IL-8 secreted by blood leukocytes stimulated by several TLR agonists. RTDs 1–5 suppressed levels of soluble TNF released by bacteria- or LPS-stimulated blood leukocytes and THP-1 monocytes. Despite their highly conserved conformation and amino acid sequences, the anti-TNF activities of RTDs 1–5 varied by as much as 10-fold. Systemically administered RTD-1 was non-toxic for BALB/c mice, and escalating intravenous doses were well tolerated and non-immunogenic in adult chimpanzees. The peptide was highly stable in serum and plasma. Single dose administration of RTD-1 at 5 mg/kg significantly improved survival of BALB/c mice with E. coli peritonitis and cecal ligation-and-puncture induced polymicrobial sepsis. Peptide treatment reduced serum levels of several inflammatory cytokines/chemokines in bacteremic animals. Collectively, these results indicate that the anti-inflammatory properties of θ-defensins in vitro and in vivo are mediated by the suppression of numerous proinflammatory cytokines and blockade of TNF release may be a primary effect.

Rhesus macaque θ-defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent role in neutrophil granule microbicidal activities

Mammalian defensins are cationic, antimicrobial peptides that play a central role in innate immunity. The peptides are composed of three structural subfamilies: α‐, β‐, and θ‐defensins. θ‐Defensins are macrocyclic octadecapeptides expressed only in Old World monkeys and Orangutans and are produced by the pair‐wise, head‐to‐tail splicing of nonapeptides derived from their respective precursors. The existence of three active θ‐defensin genes predicts that six different RTDs (1–6) are produced in this species. In this study, we isolated and quantified RTDs 1–6 from the neutrophils of 10 rhesus monkeys. RTD‐1 was the most abundant θ‐defensin, constituting ∼50% of the RTD content; total RTD content varied by as much as threefold between animals. All peptides tested were microbicidal at ∼1 μM concentrations. The contribution of θ‐defensins to macaque neutrophil antimicrobial activity was assessed by analyzing the microbicidal properties of neutrophil granule extracts after neutralizing θ‐defensin content with a specific antibody. θ‐Defensin neutralization markedly reduced microbicidal activities of the corresponding extracts. Macaque neutrophil granule extracts had significantly greater microbicidal activity than those of human neutrophils, which lack θ‐defensins. Supplementation of human granule extracts with RTD‐1 markedly increased the microbicidal activity of these preparations, further demonstrating a prominent microbicidal role for θ‐defensins.

Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30

ABSTRACT Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by ∼70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.

Rhesus macaque θ‐defensin RTD‐1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF‐κB and MAPK pathways

θ‐Defensins are pleiotropic, macrocyclic peptides that are expressed uniquely in Old World monkeys. The peptides are potent, broad‐spectrum microbicides that also modulate inflammatory responses in vitro and in animal models of viral infection and polymicrobial sepsis. θ‐Defensins suppress proinflammatory cytokine secretion by leukocytes stimulated with diverse Toll‐like receptor (TLR) ligands. Studies were performed to delineate anti‐inflammatory mechanisms of rhesus θ‐defensin 1 (RTD‐1), the most abundant θ‐defensin isoform in macaque granulocytes. RTD‐1 reduced the secretion of tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β, and IL‐8 in lipopolysaccharide (LPS)‐stimulated human blood monocytes and THP‐1 macrophages, and this was accompanied by inhibition of nuclear factor κB (NF‐κB) activation and mitogen‐activated protein kinase (MAPK) pathways. Peptide inhibition of NF‐κB activation occurred following stimulation of extracellular (TLRs 1/2 and 4) and intracellular (TLR9) receptors. Although RTD‐1 did not inhibit MAPK in unstimulated cells, it induced phosphorylation of Akt in otherwise untreated monocytes and THP‐1 cells. In the latter, this occurred within 10 min of RTD‐1 treatment and produced a sustained elevation of phosphorylated Akt (pAkt) for at least 4 h. pAkt is a negative regulator of MAPK and NF‐κB activation. RTD‐1 inhibited IκBα degradation and p38 MAPK phosphorylation, and stimulated Akt phosphorylation in LPS‐treated human primary monocytes and THP‐1 macrophages. Specific inhibition of phosphatidylinositol 3‐kinase (PI3K) blocked RTD‐1‐stimulated Akt phosphorylation and reversed the suppression of NF‐κB activation by the peptide. These studies indicate that the anti‐inflammatory properties of θ‐defensins are mediated by activation of the PI3K/Akt pathway and suppression of proinflammatory signals in immune‐stimulated cells.

High Fidelity Processing and Activation of the Human α-Defensin HNP1 Precursor by Neutrophil Elastase and Proteinase 3

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1–4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages (“folded proHNP1”) and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.

SDF2L1, a Component of the Endoplasmic Reticulum Chaperone Complex, Differentially Interacts with α-, β-, and θ-Defensin Propeptides

Mammalian defensins are cationic antimicrobial peptides that play a central role in host innate immunity and as regulators of acquired immunity. In animals, three structural defensin subfamilies, designated as α, β, and θ, have been characterized, each possessing a distinctive tridisulfide motif. Mature α- and β-defensins are produced by simple proteolytic processing of their prepropeptide precursors. In contrast, the macrocyclic θ-defensins are formed by the head-to-tail splicing of nonapeptides excised from a pair of prepropeptide precursors. Thus, elucidation of the θ-defensin biosynthetic pathway provides an opportunity to identify novel factors involved in this unique process. We incorporated the θ-defensin precursor, proRTD1a, into a bait construct for a yeast two-hybrid screen that identified rhesus macaque stromal cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a component of the endoplasmic reticulum (ER) chaperone complex, which we found to also interact with α- and β-defensins. However, analysis of the SDF2L1 domain requirements for binding of representative α-, β-, and θ-defensins revealed that α- and β-defensins bind SDF2L1 similarly, but differently from the interactions that mediate binding of SDF2L1 to pro-θ-defensins. Thus, SDF2L1 is a factor involved in processing and/or sorting of all three defensin subfamilies.