An Amyloidogenic Sequence at the N-Terminus of the Androgen Receptor Impacts Polyglutamine Aggregation

The human androgen receptor (AR) is a ligand inducible transcription factor that harbors an amino terminal domain (AR-NTD) with a ligand-independent activation function. AR-NTD is intrinsically disordered and displays aggregation properties conferred by the presence of a poly-glutamine (polyQ) sequence. The length of the polyQ sequence as well as its adjacent sequence motifs modulate this aggregation property. AR-NTD also contains a conserved KELCKAVSVSM sequence motif that displays an intrinsic property to form amyloid fibrils under mild oxidative conditions. As peptide sequences with intrinsic oligomerization properties are reported to have an impact on the aggregation of polyQ tracts, we determined the effect of the KELCKAVSVSM on the polyQ stretch in the context of the AR-NTD using atomic force microscopy (AFM). Here, we present evidence for a crosstalk between the amyloidogenic properties of the KELCKAVSVSM motif and the polyQ stretch at the AR-NTD

Chemical synthesis of transactivation domain (TAD) of tumor suppressor protein p53 by native chemical ligation of three peptide segments

Chemical composition of tumor suppressor protein p53 is altered via multiple post-translational modifications which modulate its cellular lifetime and interactions with other biomolecules. Here we report total chemical synthesis of a 61-residue form of transactivation domain (TAD) of p53 based on native chemical ligation of three peptide segments. The experiments to characterize its binding to nuclear co-activator binding domain (NCBD) of CREB-binding protein confirmed native-like induced folding upon binding to NCBD. Thus, the synthetic approach described herein can be useful for the preparation of various post-translationally modified analogues of TAD-p53 for further functional biochemical and biophysical studies.The research was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC-2016-StG, grant number 715062-HiChemSynPro)

Protein Expr Purif

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway

BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17 that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth

HIV-1 Residual Viremia Correlates with Persistent T-Cell Activation in Poor Immunological Responders to Combination Antiretroviral Therapy

BACKGROUND:The clinical significance and cellular sources of residual human immunodeficiency virus type 1 (HIV-1) production despite suppressive combination antiretroviral therapy (cART) remain unclear and the effect of low-level viremia on T-cell homeostasis is still debated. METHODOLOGY/PRINCIPAL FINDINGS:We characterized the recently produced residual viruses in the plasma and short-lived blood monocytes of 23 patients with various immunological responses to sustained suppressive cART. We quantified the residual HIV-1 in the plasma below 50 copies/ml, and in the CD14(high) CD16(-) and CD16+ monocyte subsets sorted by flow cytometry, and predicted coreceptor usage by genotyping V3 env sequences. We detected residual viremia in the plasma of 8 of 10 patients with poor CD4+ T-cell reconstitution in response to cART and in only 5 of 13 patients with good CD4+ T-cell reconstitution. CXCR4-using viruses were frequent among the recently produced viruses in the plasma and in the main CD14(high) CD16(-) monocyte subset. Finally, the residual viremia was correlated with persistent CD4+ and CD8+ T-cell activation in patients with poor immune reconstitution. CONCLUSIONS:Low-level viremia could result from the release of archived viruses from cellular reservoirs and/or from ongoing virus replication in some patients. The compartmentalization of the viruses between the plasma and the blood monocytes suggests at least two origins of residual virus production during effective cART. CXCR4-using viruses might be produced preferentially in patients on cART. Our results also suggest that low-level HIV-1 production in some patients may contribute to persistent immune dysfunction despite cART

An Amyloidogenic Sequence at the N-Terminus of the Androgen Receptor Impacts Polyglutamine Aggregation

The human androgen receptor (AR) is a ligand inducible transcription factor that harbors an amino terminal domain (AR-NTD) with a ligand-independent activation function. AR-NTD is intrinsically disordered and displays aggregation properties conferred by the presence of a poly-glutamine (polyQ) sequence. The length of the polyQ sequence as well as its adjacent sequence motifs modulate this aggregation property. AR-NTD also contains a conserved KELCKAVSVSM sequence motif that displays an intrinsic property to form amyloid fibrils under mild oxidative conditions. As peptide sequences with intrinsic oligomerization properties are reported to have an impact on the aggregation of polyQ tracts, we determined the effect of the KELCKAVSVSM on the polyQ stretch in the context of the AR-NTD using atomic force microscopy (AFM). Here, we present evidence for a crosstalk between the amyloidogenic properties of the KELCKAVSVSM motif and the polyQ stretch at the AR-NTD

CEP stable 1.6 cycle laser pulses at 1.8 \u3bcm

By using the novel approach for pulse compression that combines spectral broadening in hollow-core fiber (HCF) with linear propagation in fused silica (FS), we generate 1.6 cycle 0.24 mJ laser pulses at 1.8 \u3bcm wavelength with a repetition rate of 1 kHz. These pulses are obtained with a white light seeded optical parametric amplifier (OPA) and shown to be passively carrier envelope phase (CEP) stable.Peer reviewed: YesNRC publication: Ye

High harmonic generation with long-wavelength few-cycle laser pulses

We report the extension of hollow-core fibre pulse compression to longer wavelengths. High-energy multi-cycle infrared pulses are generated via optical parametric amplification and subsequently broadened in the fibre. 2.5-cycle pulses at the Signal wavelength (1.4 \ub5m) and 1.6-cycle pulses at the Idler wavelength (1.8 \ub5m) in the sub-millijoule regime have been generated. New compression schemes can be applied at 1.8 \ub5m and beyond. In this manner, 1.6-cycle carrier envelope phase stable pulses were generated by linear propagation in the anomalous dispersion regime of bulk glass which surprisingly enables compression below its third-order dispersion limit. Furthermore, a dispersion-free way of controlling the carrier envelope phase is demonstrated. Moreover, we experimentally confirm the increase in high-harmonic cut-off energy with driving laser wavelength and demonstrate the beneficial effect of few-cycle pulses which enable higher saturation intensities on target compared to multi-cycle pulses. It will be an ideal tool for future synthesis of isolated attosecond pulses in the sub-keV regime. With this laser source, we revealed for the first time multi-electron effects in high harmonic generation in xenon.Peer reviewed: YesNRC publication: Ye

Azaindoles as Zinc-Binding Small-Molecule Inhibitors of the JAMM Protease CSN5

Cullin-RING ligases (CRLs) represent the largest family of E3 ubiquitin ligases. CSN5 is the zinc metalloprotease subunit of the COP9 signalosome, an important regulator of CRLs. Elevated expression of CSN5 has been found in several types of cancers. Altmann and coworkers describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion through an unprecedented binding mode. Nanomolar inhibitors led to degradation of the substrate recognition subunit Skp2 and reduced the viability of HCT116 cells. The study provides a proof-of-concept for the potential of CSN5 inhibitors as anticancer agents