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Rationale: Mass spectra processing in protein hydrogen/deuterium (H/D) exchange has been remarkably improved by the introduction of fitting of the amide exchange probabilities to peptide isotopic envelope intensities (Kan et al., 2013), in contrast to methods in which only the peptide deuterium uptakes (centroid shifts of isotopic envelopes) are used. However, the known implementations are based on the general fitting routines that use only the objective function values. Besides, applicability of more than one fitting method makes necessary their comparative evaluation., Methods: Two fitting methods were considered: the common least squares and the fitting of the multinomial distribution representing the number of deuterium atoms exchanged in the individual peptides. Both methods were applied either directly to the isotopic envelope data or to the deuterium distributions obtained by envelope deconvolution (i.e. de-isotoping)., Results: An autonomous Matlab script was prepared, based on the exact expressions for the gradient and Hessian of the objective function, with the trust-region algorithm implemented in the compact analytical form recently made available. The least-squares fitting to the envelope data produced the best results, with the greatest precision and good coverage of exact values by the confidence intervals. The deuterium distributions were sensitive to the (simulated) experimental error whose progression by envelope deconvolution caused degradation in accuracy. The multinomial distribution fitting exhibited poor performance due to inadequate representation of the experimental error and missing of the appropriate weight parameters. Some specific peptide arrangement details were discussed as potential sources of ambiguity in the fitting results., Conclusions: The method of fitting to peptide isotopic envelopes has been improved by using the exact gradient and Hessian of the objective function. The fitting should be repeated with different initial guesses in order to find not only the global minimum, but also the local minima with similar depths which may exist due to eventual ambiguity of the fitting results., (© 2019 John Wiley & Sons, Ltd.)

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are related peptides with distinct pharmacological profiles. Calcitonin-receptor-like receptor (CRLR, now known as CL) can function as either an AM receptor or a CGRP receptor, when cotransfected with receptor-activity-modifying proteins (RAMPs) that define ligand-binding specificity. The aim of the present study was to determine the role of endogenously expressed CL (EndoCL) in generating endogenous AM and CGRP receptors. We raised anti-human CL antibody and identified microvascular endothelial cells (MVECs) as a major CL-expressing cell type in tissues by immunohistochemistry. Cultured MVECs continue to express EndoCL as well as fully active endogenous AM- and CGRP-sensitive receptors in vitro, as demonstrated by the ability of both peptides to induce migration and Akt phosphorylation. We therefore tested the hypothesis that endothelial EndoCL can interact with both AM and CGRP by examining receptor internalisation and desensitisation (loss of the ability to induce Akt phosphorylation). We found that agonist-mediated internalisation of EndoCL occurs in response to AM but not CGRP in MVECs. However, AM-induced EndoCL internalisation was blocked by antagonists of both AM and CGRP receptors: AM(22-52) and CGRP(8-37), respectively. Furthermore, AM-induced EndoCL internalisation resulted in desensitisation not only of AM but also of CGRP receptors. Finally, CGRP also induced desensitisation of both endogenous AM and CGRP receptors, but did not mediate EndoCL internalisation despite interaction with this receptor. Thus, EndoCL interacts with both AM and CGRP, and simultaneously acts as a receptor for both peptides (i.e acting as an endogenous AM/CGRP receptor) in endothelial cells. Interaction with either ligand is sufficient to induce EndoCL desensitisation to both AM and CGRP, but differential mechanisms are involved since only AM induces EndoCL internalisation. These novel findings regarding regulation of EndoCL function in endothelial cells are likely to be of importance in conditions where AM or CGRP levels are elevated, such as cardiovascular disease, diabetes and inflammation.

1. The calcitonin receptor-like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3. We confirmed CGRP subtype 1 receptor (CGRP(1)) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP(1) and adrenomedullin receptors whereas Rat-2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP(2) receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP(1)-like in this study. 4. L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. 5. SK-N-MC, Col-29 and Rat-2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus, circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. 6. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

The implication of reduced proteasomal function in neurodegenerative diseases combined with studies showing the protective effects of increasing proteasome activity in animal models highlight the need to understand the capacity for proteasome activation by small molecules. The C-terminal HbYX motif is present on many proteasome binding proteins and functions to tether activators to the 20S core particle. Previous studies have shown that peptides with a HbYX motif can autonomously activate 20S gate-opening to allow protein degradation. In this study, through an iterative process of peptide synthesis, we design a HbYX-like dipeptide mimetic that represents only the fundamental components of the HbYX motif. The mimetic robustly induces gate-opening in archaeal, yeast, and mammalian proteasomes. We identify multiple proteasome α subunit residues in the archaeal proteasome involved in HbYX-dependent activation. When stimulated by the mimetic, the mammalian 20S can degrade unfolded proteins such as tau. Findings using our peptide mimetic suggest the HbYX-dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Most significantly, our peptide mimetic reverses proteasome impairment by neurodegenerative disease-associated oligomers. Collectively, these results validate HbYX-like molecules as having robust potential to stimulate proteasome function, which are potentially useful for treating neurodegenerative diseases. An HbYX-like dipeptide induces gate-opening in archaeal, yeast, and mammalian 20S proteasomes, stimulates proteasome activity, and reverses inhibition of the proteasome by toxic protein oligomers that are implicated in various neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain cancers and systemic lupus erythematosus. Despite years of investigation, how PA28γ functions to stimulate proteasomal protein degradation remains unclear. Alternative hypotheses have been proposed for the molecular mechanism of PA28γ, including the following: (1) substrate selection, (2) allosteric upregulation of the trypsin-like (T-L) site, (3) allosteric inhibition of the chymotrypsin-like (CT-L) and caspase-like (C-L) sites, (4) conversion of the CT-L or C-L sites to new T-L sites, and (5) gate opening alone or in combination with a previous hypothesis. Here, by mechanistically decoupling gating effects from active site effects, we unambiguously demonstrate that WT PA28γ allosterically activates the T-L site. We show PA28γ binding increases the Kcat/Km by 13-fold for T-L peptide substrates while having little-to-no effect on hydrolysis kinetics for CT-L or C-L substrates. Furthermore, mutagenesis and domain swaps of PA28γ reveal that it does not select for T-L peptide substrates through either the substrate entry pore or the distal intrinsically disordered region. We also show that a previously reported point mutation can functionally switch PA28γ from a T-L activating to a gateopening activator in a mutually exclusive fashion. Finally, using cryogenic electron microscopy, we visualized the PA28γ-proteasome complex at 4.3 Å and confirmed its expected quaternary structure. The results of this study provide unambiguous evidence that PA28γ can function by binding the 20S proteasome to allosterically activate the T-L proteolytic site. [ABSTRACT FROM AUTHOR]

Electrically controllable deoxyribonuclic acid (DNA) nanolevers are used to investigate the binding interaction between Influenza A/Aichi/2/1968 and the peptide called "PeB", which specifically binds the viral surface protein hemagglutinin. PeB is immobilized on gold electrodes of a "switchSENSE" biochip by conjugation to DNA‐strands that are hybridized to complementary anchors. The surface‐tethered DNA strand carries a fluorophore while the complementary strand is a multivalent arrangement carrying up to three PeB peptides. The nanolevers are kept upright (static) by applying a negative potential. Signal read‐out for this static measurement mode is the change in fluorescence intensity due to changes in the local environment of the dye upon binding. Measurements of virus‐peptide interaction show that the virus material specifically binds to the immobilized peptides and remains bound throughout the measurement time. Immobilized viruses are subsequently used as ligands to characterize oligovalent peptide binding to hemagglutinin, revealing rate constants of the interaction. Moreover, three Influenza A subtypes are compared in their binding behavior. Overall, this paper shows the ability to immobilize virus material on a sensor surface, which allows to target virus‐proteins in their native environment. The "switchSENSE" method is therefore applicable to characterize virus‐receptor interactions. [ABSTRACT FROM AUTHOR]

Summary: The 20S proteasome functions in protein degradation in eukaryotes together with the 19S ATPases or in archaea with the homologous PAN ATPase complex. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X motif (HbYX). We show that these residues are essential for PAN to associate with the 20S and open its gated channel for substrate entry. Upon ATP binding, these C-terminal residues bind to pockets between the 20S''s α subunits. Seven-residue or longer peptides from PAN''s C terminus containing the HbYX motif also bind to these sites and induce gate opening in the 20S. Gate opening could be induced by C-terminal peptides from the 19S ATPase subunits, Rpt2, and Rpt5, but not by ones from PA28/26, which lack the HbYX motif and cause gate opening by distinct mechanisms. C-terminal residues in the 19S ATPases were also shown to be critical for gating and stability of 26S proteasomes. Thus, the C termini of the proteasomal ATPases function like a “key in a lock” to induce gate opening and allow substrate entry. [Copyright &y& Elsevier]

Virtually all age-related neurodegenerative diseases (NDs) can be characterized by the accumulation of proteins inside and outside the cell that are thought to significantly contribute to disease pathogenesis. One of the cell's primary systems for the degradation of misfolded/damaged proteins is the ubiquitin proteasome system (UPS), and its impairment is implicated in essentially all NDs. Thus, upregulating this system to combat NDs has garnered a great deal of interest in recent years. Various animal models have focused on stimulating 26S activity and increasing 20S proteasome levels, but thus far, none have targeted intrinsic activation of the 20S proteasome itself. Therefore, we constructed an animal model that endogenously expresses a hyperactive, open gate proteasome in Caenorhabditis elegans. The gate-destabilizing mutation that we introduced into the nematode germline yielded a viable nematode population with enhanced proteasomal activity, including peptide, unstructured protein, and ubiquitin-dependent degradation activities. We determined these nematodes showed a significantly increased lifespan and substantial resistance to oxidative and proteotoxic stress but a significant decrease in fecundity. Our results show that introducing a constitutively active proteasome into a multicellular organism is feasible and suggests targeting the proteasome gating mechanism as a valid approach for future age-related disease research efforts in mammals. [ABSTRACT FROM AUTHOR]

Adrenomedullin (AM), a potent vasoactive peptide, is elevated in certain disease states such as sepsis. Its role as a physiologically relevant peptide has been confirmed with the advent of the homozygous lethal AM peptide knockout mouse. So far, there have been few and conflicting studies which examine the regulatory role of AM at the receptor level. In this article, we discuss the few studies that have been presented on the desensitisation of AM receptors and also present novel data on the desensitisation of endogenous AM receptors in Rat-2 fibroblasts. [Copyright &y& Elsevier]