Control of Toxin-Antitoxin Systems by Proteases in Mycobacterium Tuberculosis

Toxin-antitoxin (TA) systems are small genetic elements composed of a noxious toxin and a counteracting cognate antitoxin. Although they are widespread in bacterial chromosomes and in mobile genetic elements, their cellular functions and activation mechanisms remain largely unknown. It has been proposed that toxin activation or expression of the TA operon could rely on the degradation of generally less stable antitoxins by cellular proteases. The resulting active toxin would then target essential cellular processes and inhibit bacterial growth. Although interplay between proteases and TA systems has been observed, evidences for such activation cycle are very limited. Herein, we present an overview of the current knowledge on TA recognition by proteases with a main focus on the major human pathogen Mycobacterium tuberculosis, which harbours multiple TA systems (over 80), the essential AAA + stress proteases, ClpC1P1P2 and ClpXP1P2, and the Pup-proteasome system.

Microstructure and Thermomechanical Characterization of Fe-28Mn-6Si-5Cr Shape Memory Alloy

Iron-based shape memory alloys (SMAs) have been widely studied during the last years, producing new formulations with potential applications in civil engineering. In the present paper, the microstructure and the thermomechanical behavior of the Fe-28Mn-6Si-5Cr memory alloy has been investigated. At room temperature, the presence of ε-martensite and γ-austenite was confirmed using optical and electron microscopy techniques. The martensitic transformation temperatures (As, Af, Ms, and Mf) were determined by differential scanning calorimetry, together with an X-ray diffraction technique. The use of these techniques also confirmed that this transformation is not totally reversible, depending on the strain degree and the number of thermal cycles. From the kinetics study of the ε → γ transformation, the isoconversion curves (transformation degree versus time) were built, which provided the information required to optimize the thermal activation cycle. Tensile tests were performed to characterize the mechanical properties of the studied alloy. These kinds of tests were also performed to assess the shape memory effect, getting a recovery stress of 140 MPa, after a 7.6% pre-strain and a thermal activation up to 160 °C.

Shape Memory Alloy (SMA) Actuator With Embedded Liquid Metal Curvature Sensor for Closed-Loop Control

We introduce a soft robot actuator composed of a pre-stressed elastomer film embedded with shape memory alloy (SMA) and a liquid metal (LM) curvature sensor. SMA-based actuators are commonly used as electrically-powered limbs to enable walking, crawling, and swimming of soft robots. However, they are susceptible to overheating and long-term degradation if they are electrically stimulated before they have time to mechanically recover from their previous activation cycle. Here, we address this by embedding the soft actuator with a capacitive LM sensor capable of measuring bending curvature. The soft sensor is thin and elastic and can track curvature changes without significantly altering the natural mechanical properties of the soft actuator. We show that the sensor can be incorporated into a closed-loop “bang-bang” controller to ensure that the actuator fully relaxes to its natural curvature before the next activation cycle. In this way, the activation frequency of the actuator can be dynamically adapted for continuous, cyclic actuation. Moreover, in the special case of slower, low power actuation, we can use the embedded curvature sensor as feedback for achieving partial actuation and limiting the amount of curvature change.

Exploratory experiments on pre-activated freezing nucleation on mercuric iodide

Pre-activation of freezing nucleation (PFN) with mercuric iodide was first reported by Edwards, Evans, and Zipper (Edwards et al., 1970). They found that freezing, followed by melting just a few degrees Celsius above the melting point, leads to subsequent freezing of the sample more than 10 ∘C above the temperature of the initial nucleation temperature. Results presented in this paper are from laboratory experiments that followed the procedure designed by Edwards, Evans, and Zipper (1970) but employed multiple sample drops and many repetitions of the pre-activation cycle. The results obtained confirm the basic findings of the earlier work and refine them. It is shown that the pre-activation effect is lost gradually as the sample is heated above the melting point and that some effect is still seen with heating above +5 ∘C. Instrumental limitation in these experiments precluded detection of pre-activated freezing above −2 ∘C, but that possibility is not excluded. Some PFN was noted down to at least −6 ∘C. By also drawing on the results of Seeley and Seidler (2001), PFN is analyzed in search of constraints that help define the process responsible for it. No firm conclusions are reached, but the accumulated evidence points quite clearly to the role of surface sites in leading to PFN. Thus, sites are seen to play the same role as they do in heterogeneous freezing nucleation in general. PFN differs from pore condensation and freezing described by Marcolli (2020) and David et al. (2020), in that PFN is observed in liquid water while that process takes place in the vapor phase. Further explorations of the process leading to PFN can help in understanding ice nucleation and its practical manifestations at a basic level. The results call attention to an ice nucleation pathway hitherto barely explored that can be expected to have consequences in how ice nucleation occurs in atmospheric clouds and in other systems. PFN is also a potential tool for deliberate initiation of freezing in clouds and other systems.

The Multi-Level Mechanism of Action of a Pan-Ras Inhibitor Explains its Antiproliferative Activity on Cetuximab-Resistant Cancer Cells

Ras oncoproteins play a crucial role in the onset, maintenance, and progression of the most common and deadly human cancers. Despite extensive research efforts, only a few mutant-specific Ras inhibitors have been reported. We show that cmp4–previously identified as a water-soluble Ras inhibitor– targets multiple steps in the activation and downstream signaling of different Ras mutants and isoforms. Binding of this pan-Ras inhibitor to an extended Switch II pocket on HRas and KRas proteins induces a conformational change that down-regulates intrinsic and GEF-mediated nucleotide dissociation and exchange and effector binding. A mathematical model of the Ras activation cycle predicts that the inhibitor severely reduces the proliferation of different Ras-driven cancer cells, effectively cooperating with Cetuximab to reduce proliferation even of Cetuximab-resistant cancer cell lines. Experimental data confirm the model prediction, indicating that the pan-Ras inhibitor is an appropriate candidate for medicinal chemistry efforts tailored at improving its currently unsatisfactory affinity.

Ash1 and Tup1 dependent repression of the Saccharomyces cerevisiae HO promoter requires activator-dependent nucleosome eviction

Transcriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.

GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy

In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[13CH3] labelled thermostabilized β1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with β1 adrenergic receptor (β1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tβ1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.

Exploratory experiments on pre-activated freezing nucleation on mercuric iodide

Pre-activation of freezing nucleation was examined in laboratory experiments with mercuric iodide suspensions in water. The experiments followed the procedure designed by Edwards, Evans and Zipper (1970) but employed multiple sample drops and many repetitions of the pre-activation cycle. The results obtained confirm the basic findings of the earlier work and refine it. By also drawing on the results of Seeley and Seidler (2001), pre-activated freezing nucleation (PFN in this work) is analyzed in search of constraints that help define the process responsible for it. No firm conclusions are reached, but evidence is accumulated pointing to the role of definite structures being involved in PFN, similar to the role of sites in heterogeneous freezing nucleation in general. PFN differs from pore condensation and freezing described by Marcolli (2020) and David et al. (2020) in that it takes place in liquid water. Further exploration of this process can help understading ice nucleation at the basic level and in its practical manifestations. The results call attention to an ice nucleation pathway hitherto barely explored and which can be expected to have consequences in how ice nucleation occurs in atmospheric clouds and in other systems.

Ash1 and Tup1 Dependent Repression of the Saccharomyces cerevisiae HO promoter Requires Activator-Dependent Nucleosome Eviction

ABSTRACTTranscriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.

Aha-type co-chaperones: the alpha or the omega of the Hsp90 ATPase cycle?

Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that plays an essential role in cellular homeostasis. It functions in the context of a structurally dynamic ATP-dependent cycle to promote conformational changes in its clientele to aid stability, maturation, and activation. The client activation cycle is tightly regulated by a cohort of co-chaperone proteins that display specific binding preferences for certain conformations of Hsp90, guiding Hsp90 through its functional ATPase cycle. Aha-type co-chaperones are well-known to robustly stimulate the ATPase activity of Hsp90 but other roles in regulating the functional cycle are being revealed. In this review, we summarize the work done on the Aha-type co-chaperones since the 1990s and highlight recent discoveries with respect to the complexity of Hsp90 cycle regulation.