Phillip Cruz

Evidence for Z-form RNA by vacuum UV circular dichroism

Circular dichroism (CD) spectra in the vacuum UV region for different conformations of poly d(G-C) X poly d(G-C) and poly r(G-C) X poly r(G-C) are very characteristic. The CD of the RNA in the A-form (6 M NaClO4 and 22 degrees C) is very similar to that of the DNA in 80% alcohol where it is believed to be in the A-form. With the exception of the longest wavelength transition, the CD of the RNA in 6 M NaClO4 at 46 degrees C is similar to the CD of the DNA under conditions where it is believed to be in the Z-form (2 M NaClO4). This substantiates that poly r(G-C) X poly r(G-C) assumes a left-handed Z-conformation in 6 M NaClO4 above 35 degrees C. CD spectra for the left-handed Z-forms of both the RNA and DNA are characterized by an intense negative peak at 190-195 nm, a crossover at about 184 nm, and an intense positive peak below 180 nm. The right-handed A- and B-forms of RNA and DNA all have an intense positive peak in their CD spectra near 186 nm. The large difference in CD in the range 185-195 nm for right- and left-handed conformations of nucleic acids can be used to identify the sense of helix winding.

Nearest-Neighbor Effects in the Structure and Function of Nucleic Acids

Publisher Summary This article examines the hypothesis, called the RY model, that these extended stacks occur in the special arrangements of purines (R) and pyrimidines (Y) along the chains. The RY model comes from the measurements of the nuclear magnetic resonance (nmr) chemical shifts in oligoribonucleotide and finds some support in X-ray diffraction studies. The model will evolve into a more comprehensive picture when the nearest-neighbor effects are characterized more thoroughly by further experiments. The purpose is to stimulate the molecular biologists to think beyond sequence, to consider the arrangement of nearest neighbors. It is largely these neighbor interactions that determine the local three dimensional shape imparted by the sequence. The A-form may represent the conformation of DNA in its actively expressed form. B-DNA may be a form useful for storage rather than expression of genetic content, although enzymes may also use its special plasticity in “induced-fit’’ interactions. The left-handed Z-form exists only in specific sequences and is in a delicate balance with the right-handed B-form, easily shifted by solution conditions. Z-DNA may be especially important in regulating torsional stress in supercoiled DNA.

DBAASP v.2: an enhanced database of structure and antimicrobial/cytotoxic activity of natural and synthetic peptides

Antimicrobial peptides (AMPs) are anti-infectives that may represent a novel and untapped class of biotherapeutics. Increasing interest in AMPs means that new peptides (natural and synthetic) are discovered faster than ever before. We describe herein a new version of the Database of Antimicrobial Activity and Structure of Peptides (DBAASPv.2, which is freely accessible at http://dbaasp.org). This iteration of the database reports chemical structures and empirically-determined activities (MICs, IC50, etc.) against more than 4200 specific target microbes for more than 2000 ribosomal, 80 non-ribosomal and 5700 synthetic peptides. Of these, the vast majority are monomeric, but nearly 200 of these peptides are found as homo- or heterodimers. More than 6100 of the peptides are linear, but about 515 are cyclic and more than 1300 have other intra-chain covalent bonds. More than half of the entries in the database were added after the resource was initially described, which reflects the recent sharp uptick of interest in AMPs. New features of DBAASPv.2 include: (i) user-friendly utilities and reporting functions, (ii) a ‘Ranking Search’ function to query the database by target species and return a ranked list of peptides with activity against that target and (iii) structural descriptions of the peptides derived from empirical data or calculated by molecular dynamics (MD) simulations. The three-dimensional structural data are critical components for understanding structure–activity relationships and for design of new antimicrobial drugs. We created more than 300 high-throughput MD simulations specifically for inclusion in DBAASP. The resulting structures are described in the database by novel trajectory analysis plots and movies. Another 200+ DBAASP entries have links to the Protein DataBank. All of the structures are easily visualized directly in the web browser.

Extensive synthesis of poly[r(G-C)] using Escherichia coli RNA polymerase.

Conditions have been found that allow for extensive synthesis of poly[r(G-C)] using Escherichia coli RNA polymerase with a poly[d(I-C) X poly[d(I-C)] template. The extensive synthesis, in which many copies of the template were produced by the enzyme, continued for 24 h. Repeated addition of the template was necessary for synthesis to continue, as the poly[r(G-C)] appeared to be binding to the template, inhibiting transcription by the polymerase. Four hundred ODU, or 20 mg of the poly[r(G-C)] has been prepared using 0.3 mg of protein.

Z-RNA: A Left-Handed Double Helix

The synthetic polyribonucleotide, poly [r(G-C)], has been shown to undergo a transition from the right handed A-form to a left-handed Z-conformation.1 This is the first major conformational change found in double-stranded RNA.2 Therefore it is important to corroborate this observation, to characterize the conditions that produce Z-RNA, and to determine some of the properties of this novel left-handed structure. This article is a summary of our results in this area.

Novel signal transducer and activator of transcription 1 mutation disrupts small ubiquitin-related modifier conjugation causing gain of function

Background Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin‐related modifier (SUMO) and comprises an important regulator of protein function. Objective We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1). Methods STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1‐deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs. Results We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (702IKTE705) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild‐type (WT) STAT1 cells, as well as the loss‐of‐function mutants L706S and Y701C. Furthermore, stimulation with IFN‐&ggr; led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V‐transfected compared with WT‐transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss‐of‐function and gain‐of‐function variants. Conclusion This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain‐of‐function STAT1 disease in human subjects.

A G-protein-coupled chemoattractant receptor recognizes lipopolysaccharide for bacterial phagocytosis

Phagocytes locate microorganisms via chemotaxis and then consume them using phagocytosis. Dictyostelium amoebas are stereotypical phagocytes that prey on diverse bacteria using both processes. However, as typical phagocytic receptors, such as complement receptors or Fcγ receptors, have not been found in Dictyostelium, it remains mysterious how these cells recognize bacteria. Here, we show that a single G-protein-coupled receptor (GPCR), folic acid receptor 1 (fAR1), simultaneously recognizes the chemoattractant folate and the phagocytic cue lipopolysaccharide (LPS), a major component of bacterial surfaces. Cells lacking fAR1 or its cognate G-proteins are defective in chemotaxis toward folate and phagocytosis of Klebsiella aerogenes. Computational simulations combined with experiments show that responses associated with chemotaxis can also promote engulfment of particles coated with chemoattractants. Finally, the extracellular Venus-Flytrap (VFT) domain of fAR1 acts as the binding site for both folate and LPS. Thus, fAR1 represents a new member of the pattern recognition receptors (PRRs) and mediates signaling from both bacterial surfaces and diffusible chemoattractants to reorganize actin for chemotaxis and phagocytosis.