Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61±24% decrease for cardiac tissue and 38±7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane-bound HRas decreased growth factor-stimulated ERK phosphorylation (84±8.6% decrease) and increased apoptotic signaling (24±6.5-fold increase) after HPHG treatment that was prevented by overexpressing wild-type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17-HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.     

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn(2+) and Mn(2+) cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (4(4)) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N(40) DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.     

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1_89.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.     

The physiological arms race: Exploring thermal acclimation among interacting species

Thermal acclimation is often expected to increase performance during survival-related interactions, such as prey-capture and predator escape. However, few studies have examined acclimation responses in the context of the organism's ecology: namely, considering interactions among different species. In this study, we investigated the acclimation responses of three species of aquatic organisms from the same environment that simultaneously interact across different seasons. We predicted that the crimson spotted rainbowfish (Melanotaenia duboulayi) and the freshwater shrimp (Paratya australiensis), which are involved in a predator–prey relationship, would exhibit similar thermal acclimation responses due to an arms race in physiological responses. In contrast, we expected that the backswimmer (Enithares sp.) species from the same environment, which is not commonly preyed upon due to their hard chitinous exterior, would display a limited acclimation response. We found that acute increases in temperature resulted in improved locomotor performance for all three species and an improved prey capture performance for M. duboulayi. Acclimation to 15 °C or 25 °C for 6 weeks did not affect the prey-capture performance of M. duboulayi or the locomotor performance of either M. duboulayi or Enithares sp. However, acclimation to cool temperatures improved the locomotor performance of P. australiensis at both cool and warm temperatures. Thus, the interaction between M. duboulayi (predator) and P. australiensis (prey) is likely to change across seasons via differences in acclimation responses, which could directly affect their behavioural strategies and population dynamics.     

TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site

The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ～70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ～70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ～70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.     

Isolated interstitial nodal spaces may facilitate preferential solute and fluid mixing in the rat renal inner medulla

Recent anatomic findings indicate that in the upper inner medulla of the rodent kidney, tubules, and vessels are organized around clusters of collecting ducts (CDs). Within CD clusters, CDs and some of the ascending vasa recta (AVR) and ascending thin limbs (ATLs), when viewed in transverse sections, form interstitial nodal spaces, which are arrayed at structured intervals throughout the inner medulla. These spaces, or microdomains, are bordered on one side by a single CD, on the opposite side by one or more ATLs, and on the other two sides by AVR. To study the interactions among these CDs, ATLs, and AVR, we have developed a mathematical compartment model, which simulates steady-state solute exchange through the microdomain at a given inner medullary level. Fluid in all compartments contains Na(+), Cl(-), urea and, in the microdomain, negative fixed charges that represent macromolecules (e.g., hyaluronan) balanced by Na(+). Fluid entry into AVR is assumed to be driven by hydraulic and oncotic pressures. Model results suggest that the isolated microdomains facilitate solute and fluid mixing among the CDs, ATLs, and AVR, promote water withdrawal from CDs, and consequently may play an important role in generating the inner medullary osmotic gradient.     

Light-induced plasticity of synaptic AMPA receptor composition in retinal ganglion cells

Light-evoked responses of all three major classes of?retinal ganglion cells (RGCs) are mediated by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Although synaptic activity at RGC synapses is highly dynamic, synaptic plasticity has not been observed in adult RGCs. Here, using patch-clamp recordings in dark-adapted mouse retina, we report a retina-specific form of AMPAR plasticity. Both chemical and light activation of NMDARs caused the selective endocytosis of GluA2-containing, Ca(2+)-impermeable AMPARs on RGCs and replacement with GluA2-lacking, Ca(2+)-permeable AMPARs. The plasticity was expressed in ON but not OFF RGCs and was restricted solely to the ON responses in ON-OFF RGCs. Finally, the plasticity resulted in a shift in the light responsiveness of ON RGCs. Thus, physiologically relevant light stimuli can induce a change in synaptic receptor composition of ON RGCs, providing a mechanism by which the sensitivity of RGC responses may be modified under scotopic conditions.     

CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL

The pro-apoptotic BH3 only protein BIM_EL is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM_EL to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM_EL, which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5−/− fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM_EL occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM_EL drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM_EL and may be relevant to the therapeutic use of agents such as paclitaxel.