Characterization of cooperative PS-oligo activation of human TLR9.

Single-stranded phosphorothioate oligonucleotides (PS-oligos) can activate TLR9, leading to an innate immune response. This can occur with PS-oligos containing unmethylated CpG sites, the canonical motif, or PS-oligos that do not contain those motifs (non-CpG). Structural evidence shows that TLR9 contains two PS-oligo binding sites, and recent data suggest that synergistic cooperative activation of TLR9 can be achieved by adding two separate PS-oligos to cells, each engaging with a separate site on TLR9 to enhance TLR9 activation as a pair.

Systematic Analysis of Chemical Modifications of Phosphorothioate Antisense Oligonucleotides that Modulate Their Innate Immune Response.

While rare, some gapmer phosphorothioate (PS) antisense oligonucleotides (ASOs) can induce a noncanonical TLR9-dependent innate immune response. In this study, we performed systematic analyses of the roles of PS ASO backbone chemistry, 2' modifications, and sequence in PS ASO induced TLR9 signaling. We found that each of these factors can contribute to altering PS ASO induced TLR9 signaling, and in some cases the effects are quite dramatic.

Inflammatory Non-CpG Antisense Oligonucleotides Are Signaling Through TLR9 in Human Burkitt Lymphoma B Bjab Cells.

Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability.

Insights into innate immune activation via PS-ASO-protein-TLR9 interactions.

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary-in part-as a function of 2'modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system.

The ABCs of the atypical Fam20 secretory pathway kinases.

The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome.

Gapmer Antisense Oligonucleotides Targeting 5S Ribosomal RNA Can Reduce Mature 5S Ribosomal RNA by Two Mechanisms.

In this study, we demonstrate that 5S ribosomal RNA (rRNA), a highly structured and protein-bound RNA, is quite difficult to reduce with antisense oligonucleotides (ASOs). However, we found a single accessible site that was targetable with a high-affinity complementary ASO. The ASO appeared to bind to the site, recruit RNaseH1, and cause degradation of the 5S RNA.

A secretory pathway kinase regulates sarcoplasmic reticulum Ca homeostasis and protects against heart failure.

Ca signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen.

Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia.

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores.

DNA adenine methyltransferase facilitated diffusion is enhanced by protein-DNA "roadblock" complexes that induce DNA looping.

The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein-DNA complexes (roadblocks) diminish and-interestingly-enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA adenine methyltransferase (Dam, recognizes 5'-GATC-3') with tightly bound EcoRV ENase-DNA complexes, which bend DNA.

Distinct facilitated diffusion mechanisms by E. coli Type II restriction endonucleases.

The passive search by proteins for particular DNA sequences involving nonspecific DNA is essential for gene regulation, DNA repair, phage defense, and diverse epigenetic processes. Distinct mechanisms contribute to these searches, and it remains unresolved as to which mechanism or blend of mechanisms best suits a particular protein and, more importantly, its biological role. To address this, we compare the translocation properties of two well-studied bacterial restriction endonucleases (ENases), EcoRI and EcoRV.

DNA looping provides for "intersegmental hopping" by proteins: a mechanism for long-range site localization.

Studies on how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites that controls the expression of virulence genes in several human pathogens.

Proximal recognition sites facilitate intrasite hopping by DNA adenine methyltransferase: mechanistic exploration of epigenetic gene regulation.

The methylation of adenine in palindromic 5'-GATC-3' sites by Escherichia coli Dam supports diverse roles, including the essential regulation of virulence genes in several human pathogens. As a result of a unique hopping mechanism, Dam methylates both strands of the same site prior to fully dissociating from the DNA, a process referred to as intrasite processivity. The application of a DpnI restriction endonuclease-based assay allowed the direct interrogation of this mechanism with a variety of DNA substrates.