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Betaglycan (BG) is a membrane-bound co-receptor of the TGF-β family that selectively binds transforming growth factor-β (TGF-β) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo. Here, using NMR titrations of methyl-labeled TGF-β2 with BG's C-terminal binding domain, BG(ZP-C), and surface plasmon resonance binding measurements with TGF-β2 variants, we found that the BG(ZP-C)–binding site on TGF-β2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-β isoforms and the InhA α-subunit, but they are unconserved in other TGF-β family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-β2 variants with the corresponding residues from BMP-2 bound BG(ZP-C) more weakly than corresponding alanine variants. The BG(ZP-C)–binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-β2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-β2 and InhA bind BG(ZP-C) through a site on the inside of their extended finger regions. Overall, these results identify the BG(ZP-C)–binding site on TGF-β2 and shed light on the specificity of BG for select TGF-β–type GFs and the mechanisms by which BG influences their signaling.

International audience; A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13 C− 13 C distance constraints

It has been long assumed that post-mitotic neurons only utilize the error-prone non-homologous end–joining pathway to repair double-strand breaks (DSBs) associated with oxidative damage to DNA, given the inability of non-replicating neuronal DNA to utilize a sister chromatid template in the less error-prone homologous recombination (HR) repair pathway. However, we and others have found recently that active transcription triggers a replication-independent recombinational repair mechanism in G0/G1 phase of the cell cycle. Here we observed that the HR repair protein RAD52 is recruited to sites of DNA DSBs in terminally differentiated, post-mitotic neurons. This recruitment is dependent on the presence of a nascent mRNA generated during active transcription, providing evidence that an RNA-templated HR repair mechanism exists in non-dividing, terminally differentiated neurons. This recruitment of RAD52 in neurons is decreased by transcription inhibition. Importantly, we found that high concentrations of amyloid β, a toxic protein associated with Alzheimer's disease, inhibits the expression and DNA damage response of RAD52, potentially leading to a defect in the error-free, RNA-templated HR repair mechanism. This study shows a novel RNA-dependent repair mechanism of DSBs in post-mitotic neurons and demonstrates that defects in this pathway may contribute to neuronal genomic instability and consequent neurodegenerative phenotypes such as those seen in Alzheimer's disease.

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-free apolipoproteins and regulates the generation of high density lipoproteins. Previously, we have shown that lack of Abca1 significantly increases amyloid deposition and cognitive deficits in Alzheimer's disease model mice expressing human amyloid-β protein precursor (APP). The goal of this study was to determine if ABCA1 plays a role in memory deficits caused by amyloid-β (Aβ) oligomers and examine neurite architecture of pyramidal hippocampal neurons. Our results confirm previous findings that Abca1 deficiency significantly impairs spatial memory acquisition and retention in the Morris water maze and long-term memory in novel object recognition of APP transgenic mice at a stage of early amyloid pathology. Neither test demonstrated a significant difference between Abca1ko and wild-type (WT) mice. We also examined the effect of intra-hippocampal infused Aβ oligomers on cognitive performance of Abca1ko mice, compared to control infusion of scrambled Aβ peptide. Age-matched WT mice undergoing the same infusions were also used as controls. In this model system, we found a statistically significant difference between WT and Abca1ko mice infused with scrambled Aβ, suggesting that Abca1ko mice are vulnerable to the effect of mild stresses. Moreover, examination of neurite architecture in the hippocampi revealed a significant decrease in neurite length, number of neurite segments, and branches in Abca1ko mice when compared to WT mice. We conclude that mice lacking ABCA1 have basal cognitive deficits that prevent them from coping with additional stressors, which is in part due to impairment of neurite morphology in the hippocampus.

// Tai Qin 1, 2 , Lindsey Barron 1 , Lu Xia 1, 3 , Haojie Huang 5 , Maria M. Villarreal 4 , John Zwaagstra 9 , Cathy Collins 9 , Junhua Yang 1 , Christian Zwieb 4 , Ravindra Kodali 8 , Cynthia S. Hinck 8 , Sun Kyung Kim 4 , Robert L. Reddick 6 , Chang Shu 2 , Maureen D. O’Connor-McCourt 9 , Andrew P. Hinck 8 , Lu-Zhe Sun 1, 7 1 Department of Cell Systems and Anatomy, University of Texas Health Science Center, San Antonio, TX, USA 2 Department of Vascular Surgery, Second Xiangya Hospital and Xiangya School of Medicine, Central South University, Hunan, China 3 Department of Gynecology and Obstetrics, Xiangya Hospital and Xiangya School of Medicine, Central South University, Hunan, China 4 Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA 5 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA 6 Department of Pathology, University of Texas Health Science Center, San Antonio, TX, USA 7 Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, Texas, USA 8 Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA USA 9 National Research Council Human Health Therapeutics Portfolio, Montreal, Quebec, Canada, Maureen O'Connor-McCourt is currently affiliated with Formation Biologics, Montreal, Quebec, Canada Correspondence to: Lu-Zhe Sun, email: SUNL@uthscsa.edu Andrew P. Hinck, email: ahinck@pitt.edu Keywords: TGF-β trap, RER, tumorigenesis, Pten, prostate cancer Received: August 15, 2016 Accepted: November 07, 2016 Published: November 14, 2016 ABSTRACT The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly 13C,15N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

Summary Compelling evidence links amyloid beta (Aβ) peptide accumulation in the brains of Alzheimer's disease (AD) patients with the emergence of learning and memory deficits, yet a clear understanding of the events that drive this synaptic pathology are lacking. We present evidence that neurons exposed to Aβ are unable to form new synapses, resulting in learning deficits in vivo . We demonstrate the Nogo receptor family (NgR1–3) acts as Aβ receptors mediating an inhibition of synapse assembly, plasticity, and learning. Live imaging studies reveal Aβ activates NgRs on the dendritic shaft of neurons, triggering an inhibition of calcium signaling. We define T-type calcium channels as a target of Aβ-NgR signaling, mediating Aβ's inhibitory effects on calcium, synapse assembly, plasticity, and learning. These studies highlight deficits in new synapse assembly as a potential initiator of cognitive pathology in AD, and pinpoint calcium dysregulation mediated by NgRs and T-type channels as key components. Video Abstract

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells., Huntington's disease is caused by a polyglutamine stretch expansion in the first exon of huntingtin. Here, the authors use infrared spectroscopy and solid-state NMR and show that polymorphic huntingtin exon1 fibres differ in their flanking regions but not their core polyglutamine amyloid structures.

Candidates for the toxic molecular species in the expanded polyglutamine (polyQ) repeat diseases range from various types of aggregates to "misfolded" monomers. One way to vet these candidates is to develop mutants that restrict conformational landscapes. Previously, we inserted two self-complementary β-hairpin enhancing motifs into a short polyQ sequence to generate a mutant, here called "βHP," that exhibits greatly improved amyloid nucleation without measurably enhancing β-structure in the monomer ensemble. We extend these studies here by introducing single-backbone H-bond impairing modifications αN-methyl Gln or l-Pro at key positions within βHP. Modifications predicted to allow formation of a fully H-bonded β-hairpin at the fibril edge while interfering with H-bonding to the next incoming monomer exhibit poor amyloid formation and act as potent inhibitors in trans of simple polyQ peptide aggregation. In contrast, a modification that disrupts intra-β-hairpin H-bonding within βHP, while also aggregating poorly, is ineffective at inhibiting amyloid formation in trans. The inhibitors constitute a dynamic version of the edge-protection negative design strategy used in protein evolution to limit unwanted protein aggregation. Our data support a model in which polyQ peptides containing strong β-hairpin encouraging motifs only rarely form β-hairpin conformations in the monomer ensemble, but nonetheless take on such conformations at key steps during amyloid formation. The results provide insights into polyQ solution structure and fibril formation while also suggesting an approach to the design of inhibitors of polyQ amyloid growth that focuses on conformational requirements for fibril and nucleus elongation.

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a Ki of 20–70 nm. Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.

Since early oligomeric intermediates in amyloid assembly are often transient and difficult to distinguish, characterize and quantify, the mechanistic basis of the initiation of spontaneous amyloid growth is often opaque. We describe here an approach to the analysis of the Aβ aggregation mechanism that uses Aβ-polyglutamine hybrid peptides designed to retard amyloid maturation and an adjusted thioflavin intensity scale that reveals structural features of aggregation intermediates. The results support an aggregation initiation mechanism for Aβ-polyQ hybrids, and by extension for full-length Aβ peptides, in which a modular Aβ C-terminal segment mediates rapid, non-nucleated formation of α-helical oligomers. The resulting high local concentration of tethered amyloidogenic segments within these α-oligomers facilitates transition to a β-oligomer population that, via further remodelling and/or elongation steps, ultimately generates mature amyloid. Consistent with this mechanism, an engineered Aβ C-terminal fragment delays aggregation onset by Aβ-polyglutamine peptides and redirects assembly of Aβ42 fibrils., The elucidation of amyloid nucleation mechanisms remains challenging as early oligomeric intermediates are transient and difficult to distinguish. Here the authors use Aβ- polyglutamine hybrid peptides designed to slow and limit amyloid maturation to provide insights into the structures of Aβ self-assembly intermediates.

Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile in the context of emerging cellular distress. The data provide some new candidates for the toxic species and some new reservations about more well-established candidates. Compared to other known markers of HTT toxicity, nuclear DNA damage appears to be a relatively early pathological event.

TGF-β1, TGF- β2, and TGF-β3 are 26 kDa disulfide-linked homodimeric signaling proteins. They all signal through the TGF-β type I and type II receptors, yet TGF-β2, which is well known to bind TβRII several-hundred fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for the TGF-β type III receptor (TβRIII), a membrane-anchored non-signaling receptor that potentiates the binding of TβRII. Though it is known that TβRIII has two component domains that bind TGF-β2 non-cooperatively at independent sites, the structure of these domains bound to TGF-β2 and residues responsible for specific binding are not yet known.

Aβ43, a product of the proteolysis of the amyloid precursor protein APP, is related to Aβ42 by an additional Thr residue at the C-terminus. Aβ43 is typically generated at low levels compared with the predominant Aβ42 and Aβ40 forms, but it has been suggested that this longer peptide might have an impact on amyloid-β aggregation and Alzheimer's disease that is out of proportion to its brain content. Here, we report that both Aβ42 and Aβ43 spontaneously aggregate into mature amyloid fibrils via sequential appearance of the same series of oligomeric and protofibrillar intermediates, the earliest of which appears to lack β-structure. In spite of the additional β-branched amino acid at the C-terminus, Aβ43 fibrils have fewer strong backbone H-bonds than Aβ42 fibrils, some of which are lost at the C-terminus. In contrast to previous reports, we found that Aβ43 spontaneously aggregates more slowly than Aβ42. In addition, Aβ43 fibrils are very inefficient at seeding Aβ42 amyloid formation, even though Aβ42 fibrils efficiently seed amyloid formation by Aβ43 monomers. Finally, mixtures of Aβ42 and Aβ43 aggregate more slowly than Aβ42 alone. Both in this Aβ42/Aβ43 co-aggregation reaction and in cross-seeding by Aβ42 fibrils, the structure of the Aβ43 in the product fibrils is influenced by the presence of Aβ42. The results provide new details of amyloid structure and assembly pathways, an example of structural plasticity in prion-like replication, and data showing that low levels of Aβ43 in the brain are unlikely to favorably impact the aggregation of Aβ42.

Cytochrome c (cyt-c) plays a key role in activating intrinsic apoptosis, a closely regulated cell death mechanism. Complete understanding of this process has implications for treatment of neurodegenerative diseases like Huntington's disease, as well as cancer. A pivotal signaling event in this apoptotic pathway is the peroxidation of the mitochondrial lipid cardiolipin (CL) by cyt-c. Cyt-c gains CL-specific lipid peroxidase activity by binding to CL during apoptosis, but exactly what structural changes lead to its proapoptotic activity have remained elusive. We have performed both structural and functional measurements to determine changes in structure and dynamics of cyt-c that accompany the activation of its peroxidase function upon binding to CL. Specifically, we used FTIR and multidimensional magic-angle-spinning (MAS) solid-state NMR (ssNMR) on samples mimicking proapoptotic conditions that trigger cyt-c's peroxidase activity. Comparison to FTIR and solution NMR data for the soluble protein revealed that cyt-c did not experience major unfolding upon membrane binding. We probed the protein-lipid interactions via ssNMR on both the protein and the lipids, and additionally used fluorescence quenching measurements of resonance energy transfer between the cyt-c heme and fluorescently labeled lipids. We conclude that increases in peroxidase activity of peripherally CL-bound cyt-c do not result from extensive unfolding of cyt-c. We instead propose an alternative mechanism in which localized structural changes, rather than global unfolding, in the CL-bound protein may be enough to allow for significant lipid peroxidation function to arise.

The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.