A glimpse into TriC's magic chamber of secrets.

Chaperones are important for protein folding, but visualizing this process has proven to be exceptionally difficult. In this issue of Cell, Frydman and colleagues have succeeded in watching tubulin being folded by its chaperonin TRiC at near-atomic resolution.

Forces Driving Chaperone Action.

It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client's affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins.

Thermonuclear explosions on neutron stars reveal the speed of their jets.

Relativistic jets are observed from accreting and cataclysmic transients throughout the Universe, and have a profound impact on their surroundings1,2. Despite their importance, their launch mechanism is not known. For accreting neutron stars, the speed of their compact jets can reveal whether the jets are powered by magnetic fields anchored in the accretion flow3 or in the star itself4,5, but so far no such measurements exist. These objects can show bright explosions on their surface due to unstable thermonuclear burning of recently accreted material, called type-I X-ray bursts6, during which the mass-accretion rate increases7-9. Here, we report on bright flares in the jet emission for a few minutes after each X-ray burst, attributed to the increased accretion rate. With these flares, we measure the speed of a neutron star compact jet to be v = 0.38 - 0.08 + 0.11 c , much slower than those from black holes at similar luminosities. This discovery provides a powerful new tool in which we can determine the role that individual system properties have on the jet speed, revealing the dominant jet launching mechanism.

Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme.

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

A rapidly changing jet orientation in the stellar-mass black-hole system V404 Cygni.

Powerful relativistic jets are one of the main ways in which accreting black holes provide kinetic feedback to their surroundings. Jets launched from or redirected by the accretion flow that powers them are expected to be affected by the dynamics of the flow, which for accreting stellar-mass black holes has shown evidence for precession1 due to frame-dragging effects that occur when the black-hole spin axis is misaligned with the orbital plane of its companion star2. Recently, theoretical simulations have suggested that the jets can exert an additional torque on the accretion flow3, although the interplay between the dynamics of the accretion flow and the launching of the jets is not yet understood. Here we report a rapidly changing jet orientation-on a time scale of minutes to hours-in the black-hole X-ray binary V404 Cygni, detected with very-long-baseline interferometry during the peak of its 2015 outburst. We show that this changing jet orientation can be modelled as the Lense-Thirring precession of a vertically extended slim disk that arises from the super-Eddington accretion rate4. Our findings suggest that the dynamics of the precessing inner accretion disk could play a role in either directly launching or redirecting the jets within the inner few hundred gravitational radii. Similar dynamics should be expected in any strongly accreting black hole whose spin is misaligned with the inflowing gas, both affecting the observational characteristics of the jets and distributing the black-hole feedback more uniformly over the surrounding environment5,6.

The enzyme pseudooxynicotine amine oxidase from Pseudomonas putida S16 is not an oxidase, but a dehydrogenase.

The soil-dwelling bacterium Pseudomonas putida S16 can survive on nicotine as its sole carbon and nitrogen source. The enzymes nicotine oxidoreductase (NicA2) and pseudooxynicotine amine oxidase (Pnao), both members of the flavin-containing amine oxidase family, catalyze the first two steps in the nicotine catabolism pathway. Our laboratory has previously shown that, contrary to other members of its enzyme family, NicA2 is actually a dehydrogenase that uses a cytochrome c protein (CycN) as its electron acceptor. The natural electron acceptor for Pnao is unknown; however, within the P. putida S16 genome, pnao forms an operon with cycN and nicA2, leading us to hypothesize that Pnao may also be a dehydrogenase that uses CycN as its electron acceptor. Here we characterized the kinetic properties of Pnao and show that Pnao is poorly oxidized by O2, but can be rapidly oxidized by CycN, indicating that Pnao indeed acts as a dehydrogenase that uses CycN as its oxidant. Comparing steady-state kinetics with transient kinetic experiments revealed that product release primarily limits turnover by Pnao. We also resolved the crystal structure of Pnao at 2.60 Å, which shows that Pnao has a similar structural fold as NicA2. Furthermore, rigid-body docking of the structure of CycN with Pnao and NicA2 identified a potential conserved binding site for CycN on these two enzymes. Taken together, our results demonstrate that although Pnao and NicA2 show a high degree of similarity to flavin containing amine oxidases that use dioxygen directly, both enzymes are actually dehydrogenases.

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O2-dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O2, severely limiting its nicotine-degrading activity. Instead of using O2 as an oxidant, we discovered that NicA2 donates electrons to a cytochrome c, which means that NicA2 is actually a dehydrogenase. This is surprising, as enzymes of the flavin-containing amine oxidase family were invariably thought to use O2 as an electron acceptor. Our findings establish new perspectives for engineering this potentially useful therapeutic and prompt a reconsideration of the term 'oxidase' in referring to members of the flavin-containing amine 'oxidase' family.

The reliability of a portable steam sauna pod for the whole-body passive heating of humans.

INTRODUCTION: Passive heating is receiving increasing attention within human performance and health contexts. A low-cost, portable steam sauna pod may offer an additional tool for those seeking to manipulate physiological (cardiovascular, thermoregulatory and sudomotor) and perceptual responses for improving sporting or health profiles. This study aimed to 1) report the different levels of heat stress and determine the pods' inter-unit reliability, and 2) quantify the reliability of physiological and perceptual responses to passive heating. METHOD: In part 1, five pods were assessed for temperature and relative humidity (RH) every 5 min across 70 min of heating for each of the 9 settings. In part 2, twelve males (age: 24 ± 4 years) completed two 60 min trials of passive heating (3 × 20 min at 44 °C/99% RH, separated by 1 week). Heart rate (HR), rectal (Trectal) and tympanic temperature (Ttympanic) were recorded every 5 min, thermal comfort (Tcomfort) and sensation (Tsensation) every 10 min, mean arterial pressure (MAP) at each break period and sweat rate (SR) after exiting the pod. RESULTS: In part 1, setting 9 provided the highest temperature (44.3 ± 0.2 °C) and longest time RH remained stable at 99% (51±7 min). Inter-unit reliability data demonstrated agreement between pods for settings 5-9 (intra-class correlation [ICC] >0.9), but not for settings 1-4 (ICC <0.9). In part 2, between-visits, high correlations, and low typical error of measurement (TEM) and coefficient of variation (CV) were found for Trectal, HR, MAP, SR, and Tcomfort, but not for Ttympanic or Tsensation. A peak Trectal of 38.09 ± 0.30 °C, HR of 124 ± 15 b min-1 and a sweat loss of 0.73 ± 0.33 L were reported. No between-visit differences (p > 0.05) were observed for Trectal, Ttympanic, Tsensation or Tcomfort, however HR (+3 b.min-1) and MAP (+4 mmHg) were greater in visit 1 vs. 2 (p < 0.05). CONCLUSION: Portable steam sauna pods generate reliable heat stress between-units. The highest setting (44 °C/99% RH) also provides reliable but modest adjustments in physiological and perceptual responses.

Candidate Tidal Disruption Event AT2019fdr Coincident with a High-Energy Neutrino.

The origins of the high-energy cosmic neutrino flux remain largely unknown. Recently, one high-energy neutrino was associated with a tidal disruption event (TDE). Here we present AT2019fdr, an exceptionally luminous TDE candidate, coincident with another high-energy neutrino. Our observations, including a bright dust echo and soft late-time x-ray emission, further support a TDE origin of this flare. The probability of finding two such bright events by chance is just 0.034%. We evaluate several models for neutrino production and show that AT2019fdr is capable of producing the observed high-energy neutrino, reinforcing the case for TDEs as neutrino sources.

Polyphosphate is a primordial chaperone.

Composed of up to 1,000 phospho-anhydride bond-linked phosphate monomers, inorganic polyphosphate (polyP) is one of the most ancient, conserved, and enigmatic molecules in biology. Here we demonstrate that polyP functions as a hitherto unrecognized chaperone. We show that polyP stabilizes proteins in vivo, diminishes the need for other chaperone systems to survive proteotoxic stress conditions, and protects a wide variety of proteins against stress-induced unfolding and aggregation. In vitro studies reveal that polyP has protein-like chaperone qualities, binds to unfolding proteins with high affinity in an ATP-independent manner, and supports their productive refolding once nonstress conditions are restored. Our results uncover a universally important function for polyP and suggest that these long chains of inorganic phosphate may have served as one of nature's first chaperones, a role that continues to the present day.

SERF engages in a fuzzy complex that accelerates primary nucleation of amyloid proteins.

The assembly of small disordered proteins into highly ordered amyloid fibrils in Alzheimer's and Parkinson's patients is closely associated with dementia and neurodegeneration. Understanding the process of amyloid formation is thus crucial in the development of effective treatments for these devastating neurodegenerative diseases. Recently, a tiny, highly conserved and disordered protein called SERF was discovered to modify amyloid formation in Caenorhabditis elegans and humans. Here, we use kinetics measurements and native ion mobility-mass spectrometry to show that SERF mainly affects the rate of primary nucleation in amyloid formation for the disease-related proteins Aß40 and α-synuclein. SERF's high degree of plasticity enables it to bind various conformations of monomeric Aß40 and α-synuclein to form structurally diverse, fuzzy complexes. This structural diversity persists into early stages of amyloid formation. Our results suggest that amyloid nucleation is considerably more complex than age-related conversion of Aß40 and α-synuclein into single amyloid-prone conformations.

Increased surface charge in the protein chaperone Spy enhances its anti-aggregation activity.

Chaperones are essential components of the protein homeostasis network. There is a growing interest in optimizing chaperone function, but exactly how to achieve this aim is unclear. Here, using a model chaperone, the bacterial protein Spy, we demonstrate that substitutions that alter the electrostatic potential of Spy's concave, client-binding surface enhance Spy's anti-aggregation activity. We show that this strategy is more efficient than one that enhances the hydrophobicity of Spy's surface. Our findings thus challenge the traditional notion that hydrophobic interactions are the major driving forces that guide chaperone-substrate binding. Kinetic data revealed that both charge- and hydrophobicity-enhanced Spy variants release clients more slowly, resulting in a greater "holdase" activity. However, increasing short-range hydrophobic interactions deleteriously affected Spy's ability to capture substrates, thus reducing its in vitro chaperone activity toward fast-aggregating substrates. Our strategy in chaperone surface engineering therefore sought to fine-tune the different molecular forces involved in chaperone-substrate interactions rather than focusing on enhancing hydrophobic interactions. These results improve our understanding of the mechanistic basis of chaperone-client interactions and illustrate how protein surface-based mutational strategies can facilitate the rational improvement of molecular chaperones.

Chaperone OsmY facilitates the biogenesis of a major family of autotransporters.

OsmY is a widely conserved but poorly understood 20 kDa periplasmic protein. Using a folding biosensor, we previously obtained evidence that OsmY has molecular chaperone activity. To discover natural OsmY substrates, we screened for proteins that are destabilized and thus present at lower steady-state levels in an osmY-null strain. The abundance of an outer membrane protein called antigen 43 was substantially decreased and its ß-barrel domain was undetectable in the outer membrane of an osmY-null strain. Antigen 43 is a member of the diffuse adherence family of autotransporters. Like strains that are defective in antigen 43 production, osmY-null mutants failed to undergo cellular autoaggregation. In vitro, OsmY assisted in the refolding of the antigen 43 ß-barrel domain and protected it from added protease. Finally, an osmY-null strain that expressed two members of the diffuse adherence family of autotransporters that are distantly related to antigen 43, EhaA and TibA, contained reduced levels of the proteins and failed to undergo cellular autoaggregation. Taken together, our results indicate that OsmY is involved in the biogenesis of a major subset of autotransporters, a group of proteins that play key roles in bacterial pathogenesis.

In vivo chloride concentrations surge to proteotoxic levels during acid stress.

To successfully colonize the intestine, bacteria must survive passage through the stomach. The permeability of the outer membrane renders the periplasm of Gram-negative bacteria vulnerable to stomach acid, which inactivates proteins. Here we report that the semipermeable nature of the outer membrane allows the development of a strong Donnan equilibrium across this barrier at low pH. As a result, when bacteria are exposed to conditions that mimic gastric juice, periplasmic chloride concentrations rise to levels that exceed 0.6 M. At these chloride concentrations, proteins readily aggregate in vitro. The acid sensitivity of strains lacking acid-protective chaperones is enhanced by chloride, suggesting that these chaperones protect periplasmic proteins both from acidification and from the accompanying accumulation of chloride. These results illustrate how organisms have evolved chaperones to respond to the substantial chemical threat imposed by otherwise innocuous chloride concentrations that are amplified to proteotoxic levels by low-pH-induced Donnan equilibrium effects.

Baryons in the relativistic jets of the stellar-mass black-hole candidate 4U 1630-47.

Accreting black holes are known to power relativistic jets, both in stellar-mass binary systems and at the centres of galaxies. The power carried away by the jets, and, hence, the feedback they provide to their surroundings, depends strongly on their composition. Jets containing a baryonic component should carry significantly more energy than electron-positron jets. Energetic considerations and circular-polarization measurements have provided conflicting circumstantial evidence for the presence or absence of baryons in jets, and the only system in which they have been unequivocally detected is the peculiar X-ray binary SS 433 (refs 4, 5). Here we report the detection of Doppler-shifted X-ray emission lines from a more typical black-hole candidate X-ray binary, 4U 1630-47, coincident with the reappearance of radio emission from the jets of the source. We argue that these lines arise from baryonic matter in a jet travelling at approximately two-thirds the speed of light, thereby establishing the presence of baryons in the jet. Such baryonic jets are more likely to be powered by the accretion disk than by the spin of the black hole, and if the baryons can be accelerated to relativistic speeds, the jets should be strong sources of γ-rays and neutrino emission.

Bright radio emission from an ultraluminous stellar-mass microquasar in M 31.

A subset of ultraluminous X-ray sources (those with luminosities of less than 10(40) erg s(-1); ref. 1) are thought to be powered by the accretion of gas onto black holes with masses of ∼5-20M cicled dot, probably by means of an accretion disk. The X-ray and radio emission are coupled in such Galactic sources; the radio emission originates in a relativistic jet thought to be launched from the innermost regions near the black hole, with the most powerful emission occurring when the rate of infalling matter approaches a theoretical maximum (the Eddington limit). Only four such maximal sources are known in the Milky Way, and the absorption of soft X-rays in the interstellar medium hinders the determination of the causal sequence of events that leads to the ejection of the jet. Here we report radio and X-ray observations of a bright new X-ray source in the nearby galaxy M 31, whose peak luminosity exceeded 10(39) erg s(-1). The radio luminosity is extremely high and shows variability on a timescale of tens of minutes, arguing that the source is highly compact and powered by accretion close to the Eddington limit onto a black hole of stellar mass. Continued radio and X-ray monitoring of such sources should reveal the causal relationship between the accretion flow and the powerful jet emission.

Flexible, symmetry-directed approach to assembling protein cages.

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.

Chaperone-client interactions: Non-specificity engenders multifunctionality.

Here, we provide an overview of the different mechanisms whereby three different chaperones, Spy, Hsp70, and Hsp60, interact with folding proteins, and we discuss how these chaperones may guide the folding process. Available evidence suggests that even a single chaperone can use many mechanisms to aid in protein folding, most likely due to the need for most chaperones to bind clients promiscuously. Chaperone mechanism may be better understood by always considering it in the context of the client's folding pathway and biological function.

Electrostatic interactions are important for chaperone-client interaction in vivo.

It has long been thought that chaperones are primarily attracted to their clients through the hydrophobic effect. However, in in vitro studies on the interaction between the chaperone Spy and its substrate Im7, we recently showed that long-range electrostatic interactions also play a key role. Spy functions in the periplasm of Gram-negative bacteria, which is surrounded by a permeable outer membrane. The ionic conditions in the periplasm therefore closely mimic those in the media, which allowed us to vary the ionic strength of the in vivo folding environment. Using folding biosensors that link protein folding to antibiotic resistance, we were able to monitor Spy chaperone activity in Escherichia coli in vivo as a function of media salt concentration. The chaperone activity of Spy decreased when the ionic strength of the media was increased, strongly suggesting that electrostatic forces play a vital role in the action of Spy in vivo.