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Abstract Hsp90 is a molecular chaperone of central importance for protein homeostasis in the cytosol of eukaryotic cells, with key functional and structural traits conserved from yeast to man. During evolution, Hsp90 has gained additional functional importance, leading to an increased number of interacting co-chaperones and client proteins. Here, we show that the overall conformational transitions coupled to the ATPase cycle of Hsp90 are conserved from yeast to humans, but cycle timing as well as the dynamics are significantly altered. In contrast to yeast Hsp90, the human Hsp90 is characterized by broad ensembles of conformational states, irrespective of the absence or presence of ATP. The differences in the ATPase rate and conformational transitions between yeast and human Hsp90 are based on two residues in otherwise conserved structural elements that are involved in triggering structural changes in response to ATP binding. The exchange of these two mutations allows swapping of the ATPase rate and of the conformational transitions between human and yeast Hsp90. Our combined results show that Hsp90 evolved to a protein with increased conformational dynamics that populates ensembles of different states with strong preferences for the N-terminally open, client-accepting states.

To answer NASA’s call for a sensitive X-ray observatory in the 2030s, we present the High Energy X-ray Probe (HEX-P) mission concept. HEX-P is designed to provide the required capabilities to explore current scientific questions and make new discoveries with a broadband X-ray observatory that simultaneously measures sources from 0.2 to 80 keV. HEX-P’s main scientific goals include: 1) understand the growth of supermassive black holes and how they drive galaxy evolution; 2) explore the lower mass populations of white dwarfs, neutron stars, and stellar-mass black holes in the nearby universe; 3) explain the physics of the mysterious corona, the luminous plasma close to the central engine of accreting compact objects that dominates cosmic X-ray emission; and 4) find the sources of the highest energy particles in the Galaxy. These goals motivate a sensitive, broadband X-ray observatory with imaging, spectroscopic, and timing capabilities, ensuring a versatile platform to serve a broad General Observer (GO) and Guest Investigator (GI) community. In this paper, we present an overview of these mission goals, which have been extensively discussed in a collection of more than a dozen papers that are part of this Research Topic volume. The proposed investigations will address key questions in all three science themes highlighted by Astro2020, including their associated priority areas. HEX-P will extend the capabilities of the most sensitive low- and high-energy X-ray satellites currently in orbit and will complement existing and planned high-energy, time-domain, and multi-messenger facilities in the next decade.

A fundamental goal of modern-day astrophysics is to understand the connection between supermassive black hole (SMBH) growth and galaxy evolution. Merging galaxies offer one of the most dramatic channels for galaxy evolution known, capable of driving inflows of gas into galactic nuclei, potentially fueling both star formation and central SMBH activity. Dual active galactic nuclei (dual AGNs) in late-stage mergers with nuclear pair separations 10 keV) studies offer a relatively contamination-free tool for probing the dense obscuring environments predicted to surround the majority of dual AGN in late-stage mergers. To date, only a handful of the brightest and closest systems have been studied at these energies due to the demanding instrumental requirements involved. We demonstrate the unique capabilities of HEX-P to spatially resolve the soft and - for the first time - hard X-ray counterparts of closely-separated (∼2″−5″) dual AGNs in the local Universe. By incorporating physically-motivated obscuration models, we reproduce realistic broadband X-ray spectra expected for deeply embedded accreting SMBHs. Hard X-ray spatially resolved observations of dual AGNs—accessible only to HEX-P—will hence transform our understanding of dual AGN in the nearby Universe.

There are two contradictory views of the eROSITA bubbles: either a 10 ^4 pc scale pair of giant bubbles blown by the Galactic center (GC), or a 10 ^2 pc scale local structure coincidentally located in the direction of GC. A key element of this controversy is the distance to the bubbles. Based on the 3D dust distribution in the Galactic plane, we found three isolated, distant (500–800 pc) clouds at intermediate Galactic latitudes. Their projected morphologies perfectly match the X-ray shadows on the defining features of the north eROSITA bubble, i.e., the North Polar Spur (NPS) and the Lotus Petal Cloud (LPC), indicating that both the NPS and LPC are distant, with a distance lower limit of nearly 1 kpc. In the X-ray-dark region between the NPS and LPC, we found a few polarized radio arcs and attributed them to the bubble’s shock front. These arcs match up perfectly with the outer border of the NPS and LPC and provide a way to define the bubble’s border. The border defined in this way can be well described by the line-of-sight tangent of a 3D skewed cup model rooted in the GC. We conclude that, instead of being two independent, distant features, the NPS and LPC compose a single, giant bubble, which therefore is most plausibly a 10 kpc scale bubble rooted at the GC.

The hard X-ray emission in active galactic nuclei (AGN) and black hole X-ray binaries is thought to be produced by a hot cloud of electrons referred to as the corona. This emission, commonly described by a power law with a high-energy cutoff, is suggestive of Comptonization by thermal electrons. While several hypotheses have been proposed to explain the origin, geometry, and composition of the corona, we still lack a clear understanding of this fundamental component. NuSTAR has been playing a key role improving our knowledge of X-ray coronæ thanks to its unprecedented sensitivity above 10 keV. However, these constraints are limited to bright, nearby sources. The High Energy X-ray Probe (HEX-P) is a probe-class mission concept combining high spatial resolution X-ray imaging and broad spectral coverage (0.2–80 keV) with a sensitivity superior to current facilities. In this paper, we highlight the major role that HEX-P will play in further advancing our insights of X-ray coronæ notably in AGN. We demonstrate how HEX-P will measure key properties and track the temporal evolution of coronæ in unobscured AGN. This will allow us to determine their electron distribution and test the dominant emission mechanisms. Furthermore, we show how HEX-P will accurately estimate the coronal properties of obscured AGN in the local Universe, helping address fundamental questions about AGN unification. In addition, HEX-P will characterize coronæ in a large sample of luminous quasars at cosmological redshifts for the first time and track the evolution of coronæ in transient systems in real time. We also demonstrate how HEX-P will enable estimating the coronal geometry using spectral-timing techniques. HEX-P will thus be essential to understand the evolution and growth of black holes over a broad range of mass, distance, and luminosity, and will help uncover the black holes’ role in shaping the Universe.

The solution structure, stability, and dynamics of a broadly-acting antiviral ACE2-IgG-Fc fusion protein are determined. Small chemical compounds binding to ACE2 can be used to drastically increase the thermal stability of the ACE2 domain.

Biophysical approaches reveal how a mutation in the constant light chain domain of a patient-derived antibody destabilizes the constant domain, prevents the dimerization of the light chain, and makes it more prone to proteolytic cleavage, unleashing the amyloidogenic potential of the isolated variable domain.

Fusion of ACE2 to IgM-Fc results in hexameric ACE2-IgM-Fc proteins with potent virus neutralization efficiency of patient-isolated SARS-CoV and SARS-CoV-2 variants.

Recent models suggest approximately half of all accreting supermassive black holes (SMBHs; M _BH ≳ 10 ^5 M _⊙ ) are expected to undergo intense growth phases behind Compton-thick ( N _H > 1.5 × 10 ^24 cm ^−2 ) veils of obscuring gas. However, despite being a viable source for the seeding of SMBHs, there are currently no examples known of a Compton-thick accreting intermediate-mass black hole (IMBH; M _BH ∼ 10 ^2 –10 ^5 M _⊙ ). We present a detailed X-ray spectral analysis of IC 750—the only active galactic nuclei (AGN) to date with a precise megamaser-based intermediate mass 99% confidence. Compton-thick obscuration is well-documented to impinge substantial bias on the pursuit of SMBH AGN. Our results thus provide the first indication that Compton-thick obscuration should also be properly considered to uncover and understand the IMBH population in an unbiased manner.

We propose a second-order statistic parameter ε , the relative occurrence rate between hot Jupiters (HJs) and cold Jupiters (CJs) ( ε = η _HJ / η _CJ ), to probe the migration of gas giants. Since the planet occurrence rate is the combined outcome of the formation and migration processes, a joint analysis of HJ and CJ frequency may shed light on the dynamical evolution of giant planet systems. We first investigate the behavior of ε as the stellar mass changes observationally. Based on the occurrence rate measurements of HJs ( η _HJ ) from the Transiting Exoplanet Survey Satellite survey and CJs ( η _CJ ) from the California Legacy Survey, we find a tentative trend (97% confidence) that ε drops when the stellar mass rises from 0.8 to 1.4 M _⊙ , which can be explained by different giant planet growth and disk migration timescales around different stars. We carry out planetesimal and pebble accretion simulations, both of which can reproduce the results of η _HJ , η _CJ , and ε . Our findings indicate that the classical core accretion + disk migration model can explain the observed decreasing trend of ε . We propose two ways to increase the significance of the trend and verify the anticorrelation. Future works are required to better constrain ε , especially for M dwarfs and for more massive stars.

Type Ia supernovae (SNe) remain poorly understood despite decades of investigation. Massive computationally intensive hydrodynamic simulations have been developed and run to model an ever-growing number of proposed progenitor channels. Further complicating the matter, a large number of subtypes of Type Ia SNe have been identified in recent decades. Due to the massive computational load required, inference of the internal structure of Type Ia SNe ejecta directly from observations using simulations has previously been computationally intractable. However, deep-learning emulators for radiation transport simulations have alleviated such barriers. We perform abundance tomography on 40 Type Ia SNe from optical spectra using the radiative transfer code TARDIS accelerated by the probabilistic DALEK deep-learning emulator. We apply a parametric model of potential outer ejecta structures to comparatively investigate abundance distributions and internal ionization fractions of intermediate-mass elements (IMEs) between normal and 1991T-like Type Ia SNe in the early phases. Our inference shows that the outer ejecta of 1991T-like Type Ia SNe is underabundant in the typical intermediate mass elements that heavily contribute to the spectral line formation seen in normal Type Ia SNe at early times. Additionally, we find that the IMEs present in 1991T-like Type Ia SNe are highly ionized compared to those in the normal Type Ia population. Finally, we conclude that the transition between normal and 1991T-like Type Ia SNe appears to be continuous observationally and that the observed differences come out of a combination of both abundance and ionization fractions in these SNe populations.

The two prevailing planet formation scenarios, core accretion and disk instability, predict distinct planetary mass–metallicity relations. Yet, the detection of this trend remains challenging due to inadequate data on planet atmosphere abundance and inhomogeneities in both planet and host stellar abundance measurements. Here we analyze high-resolution spectra for the host stars of 19 transiting exoplanets to derive the C, O, Na, S, and K abundances, including planetary types from cool mini-Neptunes to hot Jupiters ( T _eq ∼ 300–2700 K; planet radius ∼0.1–2 R _J ). Our Monte Carlo simulations suggest that the current data set, updated based on Welbanks et al., is unable to distinguish between a linear relation and an independent distribution model for the abundance-mass correlation for water, Na, or K. To detect a trend with strong evidence (Bayes factor > 10) at the 2 σ confidence interval, we recommend a minimum sample of 58 planets with Hubble Space Telescope measurements of water abundances coupled with [O/H] of the host stars, or 45 planets at the JWST precision. Coupled with future JWST or ground-based high-resolution data, this well-characterized sample of planets with precise host-star abundances constitute an important ensemble of planets to further probe the abundance-mass correlation.

The community agrees that Type Ia supernovae arise from carbon/oxygen white dwarfs undergoing thermonuclear runaway. However, the full progenitor system and the process that prompts the white dwarf to explode remain unknown. Most current models suggest that the white dwarf explodes because of interaction with a binary companion that may survive the process and remain within the resulting remnant of the exploded star. Furthermore, both the pre-supernova interaction process and the explosion of the primary are expected to imprint a significant departure from ordinary stellar radii and temperatures onto the secondary, making the star identifiable against the unrelated stellar population. Identification of a surviving companion inside an SN Ia remnant might confirm a specific corresponding SN Ia progenitor channel based on the identity of the companion. We conducted a surviving companion search of the Type Ia remnant SNR 0509−67.5 based in the Large Magellanic Cloud. The well-constrained distance to and foreground extinction of the Large Magellanic Cloud allow for Bayesian inference of stellar parameters with low correlation and uncertainties. We present a deep catalog of fully characterized stars interior to SNR 0509−67.5 with radii, effective temperatures, and metallicities inferred using combined Hubble Space Telescope photometric observations across multiple visits. We then compile a list of surviving companion models appropriate for the age of the remnant (roughly 400 yr after the explosion). We compare these predictions with the inferred stellar parameters and conclude that none of the stars are consistent with the predicted signatures of a surviving companion.

In celebration of the 10th anniversary of FEBS Open Bio, we spoke to some of the key figures of the journal’s genesis, development, and its future direction, and recount here their thoughts and experiences. Prof. Félix. Goñi discusses the role of the FEBS Publication Committee in the journal's beginnings, Dr Mary Purton relates her experiences as the journal's Executive Editor, Prof. László Fésüs explains how the journal developed during his tenure as Chair of the Publication Committee, and Prof. Johannes Buchner looks forward to the future of FEBS Press and academic publishing. Finally, Prof. John (Iain) Mowbray describes his “Friday afternoon thought” to start a new journal.

AL amyloidosis is caused by the accumulation of overproduced light chain (LC) fragments as fibrils in patient organs and it is the most prevalent systemic amyloidosis. Here, the authors combine biochemical and biophysical experiments to characterise the lag phase of a patient-derived truncated LC and they identify structural transitions that precede fibril formation.

Certain bovine antibodies have ultra-long long complementarity-determining regions (CDRs) that contain a knob for antigen interaction, which is connected to the antibody through a stalk. Here, the authors combine biophysical experiments and MD simulations and show that the stalk length is critical for the folding and stability of these antibodies. The authors also demonstrate that ultra-long bovine CDRs can be grafted into human antibodies, and furthermore show that de novo designed mini-domains that bind to the SARS-CoV-2 spike protein with high affinity can be integrated as a knob in ultra-long CDRs in bovine and human antibodies, which neutralize SARS-CoV-2.

Small heat shock proteins (sHsps) form large spherical assemblies and their regulation is not well understood. Here, the authors provide insights into the mechanism of Hsp26 activation by characterising phospho-mimetic mutants of yeast Hsp26. They present cryo-EM structures of the wild-type Hsp26 40mer and its phospho-mimetic mutants that reveal the location of the thermosensor in the oligomer, and the authors also show that the thermosensor domain is targeted by phosphorylation, which relieves the intrinsic inhibition of chaperone activity.

Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.

p23 is a co-chaperone of Hsp90 but its mode of action is mechanistically not well understood. Here, the authors combine in vitro and yeast in vivo assays, biochemical measurements and NMR experiments to characterize p23 and identify two conserved helical elements in the intrinsically disordered C-terminal tail of p23 that together with the folded domain of p23 regulate the Hsp90 ATPase activity and affect the binding and maturation of Hsp90 clients.

Noji et al. test link between protein folding and misfolding upon heating and agitation. They show that folding and amyloid formation are separated by the supersaturation barrier of a protein, breakdown of which shifts the protein to the amyloid pathway. This study is useful to the field of protein folding versus self-assembly and amyloidogenesis.

The chaperone Hsp90 uses the free energy from ATP hydrolysis to control the folding of client proteins in eukaryotic cells. Here the authors provide mechanistic insights into how its catalytic activity is coupled to conformational changes by combining large-scale molecular simulations with NMR, FRET and SAXS experiments.

Methylation of a lysine residue in Hsp90 is a recently discovered post-translational modification but the mechanistic effects of this modification have remained unknown so far. Here the authors combine biochemical and biophysical approaches, molecular dynamics (MD) simulations and functional experiments with yeast and show that this lysine is a switch point, which specifically modulates conserved Hsp90 functions including co-chaperone regulation and client activation.

The eighteenth data release (DR18) of the Sloan Digital Sky Survey (SDSS) is the first one for SDSS-V, the fifth generation of the survey. SDSS-V comprises three primary scientific programs or “Mappers”: the Milky Way Mapper (MWM), the Black Hole Mapper (BHM), and the Local Volume Mapper. This data release contains extensive targeting information for the two multiobject spectroscopy programs (MWM and BHM), including input catalogs and selection functions for their numerous scientific objectives. We describe the production of the targeting databases and their calibration and scientifically focused components. DR18 also includes ∼25,000 new SDSS spectra and supplemental information for X-ray sources identified by eROSITA in its eFEDS field. We present updates to some of the SDSS software pipelines and preview changes anticipated for DR19. We also describe three value-added catalogs (VACs) based on SDSS-IV data that have been published since DR17, and one VAC based on the SDSS-V data in the eFEDS field.

S. cerevisiae encodes two Hsp90 isoforms, the constitutively expressed Hsc82 and stress-inducible Hsp82 that are 97% identical. Here, the authors combine a range of biophysical methods and show that they differ in their enzymatic properties, resilience to stress and client range, which suggests that they evolved to provide fine-tuned chaperone assistance under physiological and stress conditions.

Summary: Heat shock protein 90 (Hsp90) is a molecular chaperone regulating the activity of diverse client proteins together with a plethora of different co-chaperones. Whether these functionally cooperate has remained enigmatic. We analyze all double mutants of 11 Saccharomyces cerevisiae Hsp90 co-chaperones in vivo concerning effects on cell physiology and the activation of specific client proteins. We find that client activation is supported by a genetic network with weak epistasis between most co-chaperones and a few modules with strong genetic interactions. These include an epistatic module regulating protein translation and dedicated epistatic networks for specific clients. For kinases, the bridging of Hsp70 and Hsp90 by Sti1/Hop is essential for activation, whereas for steroid hormone receptors, an epistatic module regulating their dwell time on Hsp90 is crucial, highlighting the specific needs of different clients. Thus, the Hsp90 system is characterized by plastic co-chaperone networks fine-tuning the conformational processing in a client-specific manner.

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC’s VL domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in E. coli. We show that proteolytic cleavage of the patient LC resulting in an isolated VL domain is essential for fibril formation. Out of 11 mutations in the patient VL, only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of VL more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic.

The heat shock protein 90 (Hsp90) chaperone undergoes large conformational changes during its functional cycle. Here the authors combine in vivo, biochemical, biophysical and computational approaches and provide insights into the allosteric regulation of Hsp90 by identifying and characterizing a switch point in the Hsp90 middle domain.

Abstract Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.

Summary: Life is resilient because living systems are able to respond to elevated temperatures with an ancient gene expression program called the heat shock response (HSR). In yeast, the transcription of hundreds of genes is upregulated at stress temperatures. Besides stress protection conferred by chaperones, the function of the majority of the upregulated genes under stress has remained enigmatic. We show that those genes are required to directly counterbalance increased protein turnover at stress temperatures and to maintain the metabolism. This anaplerotic reaction together with molecular chaperones allows yeast to efficiently buffer proteotoxic stress. When the capacity of this system is exhausted at extreme temperatures, aggregation processes stop translation and growth pauses. The emerging concept is that the HSR is modular with distinct programs dependent on the severity of the stress. : Mühlhofer et al. show that under mild and severe heat stress, proteins lost due to increased aggregation and degradation are replenished by increased protein synthesis to ensure a constant pool of proteins, together with the molecular chaperones. The heat shock response is activated in modules by transcriptional regulation. Keywords: heat shock response, protein aggregation, chaperones, transcriptome, translatome, proteome, S. cerevisiae, ribosome profiling, mass spectrometry

To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc), the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG). Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the β-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that VL fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS), using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies FcγRs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual FcγRs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different FcγRs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fcγ-receptors, with the exception of high affinity FcγRI. The FcγRII and FcγRIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the FcγR binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to FcγRIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the decreased affinity of target bound antibody towards FcRs, showing the importance of the IC-formation for the FcR- mediated effector functions.

DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a Cys(4)-Cys(2)-His-Cys (C4-C2HC) tandem zinc binding motif. DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain.Here, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501-544 determined by NMR spectroscopy. The structure adopts a ββα fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners (viral and cellular proteins) seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins.