The chemokines CCL5 and CXCL12 exhibit high-affinity binding to N-terminal peptides of the non-cognate receptors CXCR4 and CCR5, respectively.

CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors.

Importin-Mediated Pathological Tau Nuclear Translocation Causes Disruption of the Nuclear Lamina, TDP-43 Mislocalization and Cell Death.

Tau is a cytosolic protein that has also been observed in the nucleus, where it has multiple proposed functions that are regulated by phosphorylation. However, the mechanism underlying the nuclear import of tau is unclear, as is the contribution of nuclear tau to the pathology of tauopathies. We have previously generated a pathological form of tau, PH-tau (pseudophosphorylation mutants S199E, T212E, T231E, and S262E) that mimics AD pathological behavior in cells, , and a mouse model.

Multiple binding modes of an N-terminal CCR5-peptide in complex with HIV-1 gp120.

The N-terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high-affinity binding to gp120. In the present study, the interactions of gp120 with a 27-residue N-terminal CCR5 peptide sulphated at position Y10 and Y14, i.e.

Expression, Purification and Refolding of a Human Na1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties.

The voltage-gated sodium channel Na1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity.

Adjuvant Radiation Therapy for Clinical Stage III Melanoma in the Modern Therapeutic Era.

Background: Adjuvant radiation therapy (RT) can decrease lymph node basin (LNB) recurrences in patients with clinically evident melanoma lymph node (LN) metastases following lymphadenectomy, but its role in the era of modern systemic therapies (ST), immune checkpoint or BRAF/MEK inhibitors, is unclear.

Patients And Methods: Patients at four institutions who underwent lymphadenectomy (1/1/2010-12/31/2019) for clinically evident melanoma LN metastases and received neoadjuvant and/or adjuvant ST with RT, or ST alone, but met indications for RT, were identified. Comparisons were made between ST alone and ST/RT groups.

The methyl C-edited/C-filtered transferred NOE for studying protein interactions with short linear motifs.

Many proteins interact with their ligand proteins by recognition of short linear motifs that are often intrinsically disordered. These interactions are usually weak and are characterized by fast exchange. NMR spectroscopy is a powerful tool to study weak interactions.

Allovalency observed by transferred NOE: interactions of sulfated tyrosine residues in the N-terminal segment of CCR5 with the CCL5 chemokine.

The N-terminal segment of the chemokine receptor Human CC chemokine receptor 5 (CCR5), Nt-CCR5, contains four tyrosine residues, Y3, Y10, Y14, and Y15. Sulfation of at least two of these tyrosine residues was found to be essential for high-affinity binding of CCR5 to its chemokine ligands. Here, we show that among the monosulfated Nt-CCR5(8-20) peptide surrogates (sNt-CCR5) those sulfated at Y15 and Y14 have the highest affinity for the CC chemokine ligand 5 (CCL5) chemokine in comparison with monosulfation at position Y10.

Normal and Pathological Tau Uptake Mediated by M1/M3 Muscarinic Receptors Promotes Opposite Neuronal Changes.

The microtubule associated protein tau is mainly found in the cell's cytosol but recently it was also shown in the extracellular space. In neurodegenerative diseases, like Alzheimer's disease (AD), pathological tau spreads from neuron to neuron enhancing neurodegeneration. Here, we show that HEK293 cells and neurons in culture uptake extracellular normal and pathological Tau.

Hyperphosphorylation of Tau Associates With Changes in Its Function Beyond Microtubule Stability.

Tau is a neuronal microtubule associated protein whose main biological functions are to promote microtubule self-assembly by tubulin and to stabilize those already formed. Tau also plays an important role as an axonal microtubule protein. Tau is an amazing protein that plays a key role in cognitive processes, however, deposits of abnormal forms of tau are associated with several neurodegenerative diseases, including Alzheimer disease (AD), the most prevalent, and Chronic Traumatic Encephalopathy (CTE) and Traumatic Brain Injury (TBI), the most recently associated to abnormal tau.

Effects of chelator lipids, paramagnetic metal ions and trehalose on liposomes by solid-state NMR.

The effects of various lipid bound paramagnetic metal ions on liposomes prepared in the presence of trehalose and chelator lipids are evaluated to observe site-specific signal changes on liposome samples with optimal resolution in solid-state NMR spectroscopy. We found that Mn, Gd and Dy have different influences on the lipid C sites depending on their penetration depths into the bilayer, which can be extracted as distance information. The trehalose-liposome mixture is efficiently packed into solid-state NMR rotors and provides optimal resolution at reasonable instrument temperatures (10-50 °C).

Our Tau Tales from Normal to Pathological Behavior.

The microtubule associated protein tau in a hyperphosphorylated form was identified as the building block of the filamentous aggregates found in the neurons of Alzheimer's disease (AD) patients. In the abnormal state, hyperphosphorylated tau from AD brains (AD P-tau) was unable to promote microtubule assembly and more importantly, it could inhibit the normal activity of tau and other MAPs. AD P-tau was able to disrupt preformed microtubules and, by binding to normal tau, turn the latter into an AD P-tau like molecule.

Liposomal TriCurin, A Synergistic Combination of Curcumin, Epicatechin Gallate and Resveratrol, Repolarizes Tumor-Associated Microglia/Macrophages, and Eliminates Glioblastoma (GBM) and GBM Stem Cells.

Glioblastoma (GBM) is a deadly brain tumor with a current mean survival of 12-15 months. Despite being a potent anti-cancer agent, the turmeric ingredient curcumin (C) has limited anti-tumor efficacy in vivo due to its low bioavailability. We have reported earlier a strategy involving the use two other polyphenols, epicatechin gallate (E) from green tea and resveratrol (R) from red grapes at a unique, synergistic molar ratio with C (C:E:R: 4:1:12.

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates.

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D.

Molecular mechanism of prion-like tau-induced neurodegeneration.

Introduction: Accumulation of hyperphosphorylated tau and the disruption of microtubules are correlated with synaptic loss and pathology of Alzheimer's disease (AD). Impaired cognitive function and pathology of AD is correlated with this lesion. This review looks at the mechanism of neurodegeneration, the prion-like behavior of tau in its interaction with normal MAPs in correlation with tau hyperphosphorylation.

Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second, and third transmembrane (TM) domains and the contiguous loops of the α-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3, was investigated by solution NMR in trifluoroethanol/water. TM1-TM3 contains helical TM domains at the predicted locations, supported by continuous sets of medium-range NOEs.

Tau-induced neurodegeneration: mechanisms and targets.

The accumulation of hyperphosphorylated tau is a common feature of several dementias. Tau is one of the brain microtubule-associated proteins. Here we discuss tau's functions in microtubule assembly and stabilization and with regard to its interactions with other proteins.

Invited review: GPCR structural characterization: Using fragments as building blocks to determine a complete structure.

The structural characterization of G protein-coupled receptors has surged since the development of methodologies to facilitate the crystallization of these highly helical, seven transmembrane, integral membrane receptors. In the past seven years, eighteen GPCR structures were determined by X-ray crystallography. The crystal structures represent a static picture of these conformationally flexible signal transducers.

Guided reconstitution of membrane protein fragments.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs.

Large multiple transmembrane domain fragments of a G protein-coupled receptor: biosynthesis, purification, and biophysical studies.

To conduct biophysical analyses on large domains of GPCRs, multimilligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of four transmembrane and five transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G(31) -A(198) ] (4TMN), four C-terminal Ste2p[T(155) -L(340) ] (4TMC), or five C-terminal Ste2p[I(120) -L(340) ] (5TMC) transmembrane segments of Ste2p.

Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor.

Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p.

Biosynthesis of peptide fragments of eukaryotic GPCRs in Escherichia coli by directing expression into inclusion bodies.

Biosynthesis of peptides in heterologous systems is often a prerequisite to biophysical analyses. Large amounts of peptides, in particular portions of membrane proteins, are needed to optimize conditions for secondary and tertiary structure analysis. Chemical synthesis of these peptides is limited by their high hydrophobicity and also due to the need to incorporate isotopic labels for high resolution NMR analysis.

Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.

The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for alpha-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [(15)N], [(15)N, (13)C], [(15)N, (13)C, (2)H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei.

Expression and biophysical analysis of two double-transmembrane domain-containing fragments from a yeast G protein-coupled receptor.

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage.