Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron-Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the Cα-helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 µs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

NeoR, a near-infrared absorbing rhodopsin.

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λmax = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λmax = 690 nm) highly fluorescent state (QF = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors.

A family of hyperpolarization-activated channels selective for protons.

Proton (H+) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few proton channels have been identified so far. Here, we identify a family of voltage-gated "pacemaker" channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpolarization and conducts protons into the cytosol. Surprisingly, protons permeate through the channel's voltage-sensing domain, whereas the pore domain is nonfunctional. Key to proton permeation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional domains, HCNL1 evolutionarily adapted to a low-Na+ freshwater environment to conserve sperm's ability to depolarize.

Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation.

Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra-sensitivity are ill-defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000-fold more abundant than the free cellular messengers cAMP, cGMP, H+ , and Ca2+ . Opto-chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP-gated channel that serves as a perfect chemo-electrical transducer. cGMP is rapidly hydrolyzed, possibly via "substrate channeling" from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate-detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification-few enzyme molecules process many messenger molecules-does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.

The impact of aortic valve replacement on survival in patients with normal flow low gradient severe aortic stenosis: a propensity-matched comparison.

AIMS: To assess the survival benefit of aortic valve replacement (AVR) in patients with normal flow low gradient severe aortic stenosis (AS). METHODS AND RESULTS: A retrospective study of prospectively collected data of 276 patients (mean age 75 ± 15 years, 51% male) with normal transaortic flow [flow rate (FR) ≥200 mL/s or stroke volume index (SVi) ≥35 mL/m2] and severe AS (aortic valve area <1.0 cm2). The outcome measure was all-cause mortality. Of the 276 patients, 151 (55%) were medically treated, while 125 (45%) underwent an AVR. Over a mean follow-up of 3.2 ± 1.8 years (range 0-6.9 years), a total of 96 (34.8%) deaths occurred: 17 (13.6%) in AVR group vs. 79 (52.3%) in those medically treated, when transaortic flow was defined by FR (P < 0.001). When transaortic flow was defined by SVi, a total of 79 (31.3%) deaths occurred: 18 (15.1%) in AVR group vs. 61 (45.9%) in medically treated (P < 0.001). In a propensity-matched multivariable Cox regression analysis adjusting for age, gender, body surface area, smoking, hypertension, diabetes mellitus, atrial fibrillation, peripheral vascular disease, chronic kidney disease, left ventricular ejection fraction, left ventricular mass, and mean aortic gradient, not having AVR was associated with a 6.3-fold higher hazard ratio (HR) of all-cause mortality [HR 6.28, 95% confidence interval (CI) 3.34-13.16; P < 0.001] when flow was defined by FR. In the SVi-guided model, it was 3.83-fold (HR 3.83, 95% CI 2.30-6.37; P < 0.001). CONCLUSION: In patients with normal flow low gradient severe AS, AVR was associated with a significantly improved survival compared with those who received standard medical treatment.

Plasma Amino Acids and Incident Type 2 Diabetes in Patients With Coronary Artery Disease.

OBJECTIVE: Altered plasma amino acid levels have been implicated as markers of risk for incident type 2 diabetes; however, amino acids are also related to established diabetes risk factors. Therefore, potential for confounding and the impact from competing risks require evaluation. RESEARCH DESIGN AND METHODS: We prospectively followed 2,519 individuals with coronary artery disease but without diabetes. Mixed Gaussian modeling identified potential for confounding. Confounding, defined as a change in effect estimate (≥10%), was investigated by comparing amino acid-incident diabetes risk in a Cox model containing age and sex with that in models adjusted for potential confounders (BMI, estimated glomerular filtration rate, HDL cholesterol, triacylglycerol, C-reactive protein), which were further adjusted for plasma glucose, competing risks, and multiple comparisons (false discovery rate = 0.05, Benjamini-Hochberg method). Finally, component-wise likelihood-based boosting analysis including amino acids and confounders was performed and adjusted for competing risks in order to identify an optimal submodel for predicting incident diabetes. RESULTS: The mean age of the source population was 61.9 years; 72% were men. During a median follow-up of 10.3 years, 267 incident cases of diabetes were identified. In age- and sex-adjusted models, several amino acids, including the branched-chain amino acids, significantly predicted incident diabetes. Adjustment for confounders, however, attenuated associations. Further adjustment for glucose and multiple comparisons rendered only arginine significant (hazard ratio/1 SD 1.21 [95% CI 1.07-1.37]). The optimal submodel included arginine and asparagine. CONCLUSIONS: Adjustment for relevant clinical factors attenuated the amino acid-incident diabetes risk. Although these findings do not preclude the potential pathogenic role of other amino acids, they suggest that plasma arginine is independently associated with incident diabetes. Both arginine and asparagine were identified in an optimal model for predicting new-onset type 2 diabetes.

Kinetic and photonic techniques to study chemotactic signaling in sea urchin sperm.

Sperm from sea urchins are attracted by chemical cues released by the egg-a mechanism called chemotaxis. We describe here the signaling pathway and molecular components endowing sperm with single-molecule sensitivity. Chemotactic signaling and behavioral responses occur on a timescale of a few milliseconds to seconds. We describe the techniques and chemical tools used to resolve the signaling events in time. The techniques include rapid-mixing devices, rapid stroboscopic microscopy, and photolysis of caged second messengers and chemoattractants.

Exercise Treadmill Testing in Moderate or Severe Aortic Stenosis: The Left Ventricular Correlates of an Exaggerated Blood Pressure Rise.

Background Exaggerated blood pressure response during exercise predicts future hypertension and cardiovascular events in general population and different patients groups. However, its clinical and prognostic implications in patients with aortic stenosis have not been previously evaluated. Methods and Results We retrospectively studied 301 patients with moderate to severe asymptomatic aortic stenosis (aged 65±12 years) who underwent echocardiography and a modified Bruce exercise treadmill test. An exaggerated blood pressure response was defined as peak systolic blood pressure ≥190 mm Hg. An abnormal blood pressure response (either blunted or exaggerated) was found in 58% of patients and abnormal left ventricular geometry in 82%. There was no difference in the rates of abnormal blood pressure responses between patients with moderate and severe aortic stenosis ([exaggerated blood pressure response: 21% versus 22%, P=0.876] and [blunted blood pressure response: 35% versus 40%, P=0.647]). Patients with exaggerated blood pressure response (21%) were more likely to be older, have hypertension, higher pretest systolic blood pressure, left ventricular ejection fraction and mass, and increased arterial stiffness (all P<0.05). In a multivariate logistic regression analysis, an exaggerated blood pressure response was associated with higher pulse pressure/stroke volume index (odds ratio 2.45, 95% confidence interval 1.02-6.00, P=0.037) and left ventricular mass (odds ratio 2.04, 95% confidence interval 1.23-3.38, P=0.012) independent of age, hypertension, aortic annulus and left atrium diameter, and left ventricular ejection fraction. Conclusions In those with aortic stenosis, exaggerated blood pressure was strongly related to higher resting blood pressure values, left ventricular mass, and increased arterial stiffness independent of hypertension.