Investigating the role of phenylalanine residues for amyloid formation of the neuropeptide neurokinin B.

Neurokinin B (NKB) is a tachykinin peptide that has diverse roles in biology, including in human reproductive development. Cellular processing of this peptide is thought to involve formation of a dense core vesicle during transit through the regulated secretory pathway. The ability of NKB to rapidly form an amyloid can contribute to formation of the secretory granule but features that support amyloid formation of NKB are not well understood. NKB contains a diphenylalanine sequence well recognised as an important motif for self-assembly of other peptides including amyloid ß. Using mutations of the diphenylalanine motif we show that this motif in NKB is necessary for amyloid formation, and it is the unique combination of aromaticity and hydrophobicity of phenylalanine that is crucial for aggregation. Using disulfide cross-linking we propose that phenylalanine at sequence position 6 is important for stabilising inter-sheet interactions in the NKB amyloid fibril. Although having a highly conserved sequence, the NKB peptide from zebrafish only contains a single phenylalanine and does not fibrillise as extensively as mammalian NKB. Analysis of self-assembly of NKB-like peptides from different species may help in elucidating their biological roles. Taken together, this work shows that mammalian NKB has evolved, within only 10 residues, a sequence optimised for rapid self-assembly, whilst also containing residues for metal-binding, receptor binding and receptor discrimination.

Spectral Phasor Analysis of Nile Red Identifies Membrane Microenvironment Changes in the Presence of Amyloid Peptides.

The interaction of protein and peptide amyloid oligomers with membranes is thought to be one of the mechanisms contributing to cellular toxicity. However, techniques to study these interactions in the complex membrane environment of live cells are lacking. Spectral phasor analysis is a recently developed biophysical technique that can enable visualisation and analysis of membrane-associated fluorescent dyes. When the spectral profile of these dyes changes as a result of changes to the membrane microenvironment, spectral phasor analysis can localise those changes to discrete membrane regions. In this study, we investigated whether spectral phasor analysis could detect changes in the membrane microenvironment of live cells in the presence of fibrillar aggregates of the disease-related Aß42 peptide or the functional amyloid neurokinin B. Our results show that the fibrils cause distinct changes to the microenvironment of nile red associated with both the plasma and the nuclear membrane. We attribute these shifts in nile red spectral properties to changes in membrane fluidity. Results from this work suggest that cells have mechanisms to avoid or control membrane interactions arising from functional amyloids which have implications for how these peptides are stored in dense core vesicles. Furthermore, the work highlights the utility of spectral phasor analysis to monitor microenvironment changes to fluorescent probes in live cells.

Essential Role of Histidine for Rapid Copper(II)-Mediated Disassembly of Neurokinin B Amyloid.

Neurokinin B is a tachykinin peptide involved in a diverse range of neuronal functions. It rapidly forms an amyloid, which is considered physiologically important for efficient packing into dense core secretory vesicles within hypothalamic neurons. Disassembly of the amyloid is thought to require the presence of copper ions, which interact with histidine at the third position in the peptide sequence. However, it is unclear how the histidine is involved in the amyloid structure and why copper coordination can trigger disassembly. In this work, we demonstrate that histidine contributes to the amyloid structure via π-stacking interactions with nearby phenylalanine residues. The ability of neurokinin B to form an amyloid is dependent on any aromatic residue at the third position in the sequence; however, only the presence of histidine leads to both amyloid formation and rapid copper-induced disassembly.

Use of long-acting injectable antiretroviral agents for human immunodeficiency Virus: A review.

The development of potent antiretroviral drugs has significantly reduced morbidity and mortality associated with human immunodeficiency virus infection, however, the effectiveness of these medications depends upon consistent daily oral intake. Non-adherence can lead to the emergence of resistance, treatment failure and disease progression. This has necessitated the development of long-acting antiretroviral formulations administrable via an infrequent dosing regimen. Long-acting injectable forms of cabotegravir and rilpivirine have reached various stages in clinical trials both for the treatment and prevention of HIV. Other long-acting agents are at various stages of development. This review evaluates the current research on the development of long-acting injectable antiretroviral agents for the treatment and prevention of HIV.

Correction: Peacey, L., et al. Copper(II) Binding by the Earliest Vertebrate Gonadotropin-Releasing Hormone, the Type II Isoform, Suggests an Ancient Role for the Metal. Int. J. Mol. Sci. 2020, 21, 7900.

The authors wish to make the following correction to this paper [...].

Copper(II) Binding by the Earliest Vertebrate Gonadotropin-Releasing Hormone, the Type II Isoform, Suggests an Ancient Role for the Metal.

In vertebrate reproductive biology copper can influence peptide and protein function both in the pituitary and in the gonads. In the pituitary, copper binds to the key reproductive peptides gonadotropin-releasing hormone I (GnRH-I) and neurokinin B, to modify their structure and function, and in the male gonads, copper plays a role in testosterone production, sperm morphology and, thus, fertility. In addition to GnRH-I, most vertebrates express a second isoform, GnRH-II. GnRH-II can promote testosterone release in some species and has other non-reproductive roles. The primary sequence of GnRH-II has remained largely invariant over millennia, and it is considered the ancestral GnRH peptide in vertebrates. In this work, we use a range of spectroscopic techniques to show that, like GnRH-I, GnRH-II can bind copper. Phylogenetic analysis shows that the proposed copper-binding ligands are retained in GnRH-II peptides from all vertebrates, suggesting that copper-binding is an ancient feature of GnRH peptides.

Endocytic recycling prevents copper accumulation in astrocytoma cells stimulated with copper-bound neurokinin B.

Neurokinin B (NKB) is a key neuropeptide in reproductive endocrinology where it contributes to the generation of pulses of gonadotropin-releasing hormone. NKB is a copper-binding peptide; in the absence of metal NKB rapidly adopts an amyloid structure, but copper binding inhibits amyloid formation and generates a structure that can activate the neurokinin 3 receptor. The fate of copper once it binds NKB and activates the neurokinin 3 receptor is not understood, but endocytosis of NKB occurs even when the peptide is coordinated to copper. Using astrocytoma cells that express endogenous neurokinin 3 receptor, this work shows that endocytosis of apo- and copper-bound NKB occurs in concert with the receptor via a trafficking pathway that includes the early endosome. When cells are stimulated with copper-bound NKB the cellular copper concentration does not significantly increase, however when the cells are pre-treated with the recycling inhibitor, brefeldin A, they are capable of accumulating copper. This data shows that copper-bound NKB can activate the neurokinin 3 receptor then endocytosis abstracts metal, peptide and receptor from the cell surface. The cell does not accumulate the copper but instead it enters recycling pathways that ultimately leads to metal release from the cell. The work reveals a novel receptor-mediated copper trafficking pathway that retains metal in membrane bound organelles until it is exported from the cell.

Roles of copper in neurokinin B and gonadotropin-releasing hormone structure and function and the endocrinology of reproduction.

Copper is a metal ion present in all organisms, where it has well-known roles in association with proteins and enzymes essential for cellular processes. In the early decades of the twentieth century copper was shown to influence mammalian reproductive biology, and it was subsequently shown to exert effects primarily at the level of the pituitary gland and/or hypothalamic regions of the brain. Furthermore, it has been reported that copper can interact with key neuropeptides in the hypothalamic-pituitary-gonadal axis, notably gonadotropin-releasing hormone (GnRH) and neurokinin B. Interestingly, recent phylogenetic analysis of the sequences of GnRH-related peptides indicates that copper binding is an evolutionarily ancient property of this neuropeptide family, which has been variously retained, modified or lost in the different taxa. In this mini-review the metal-binding properties of neuropeptides in the vertebrate reproductive pathway are reviewed and the evolutionary and functional significance of copper binding by GnRH-related neuropeptides in vertebrates and invertebrates are discussed.

HIV-1 Accessory Protein Vpr Interacts with REAF/RPRD2 To Mitigate Its Antiviral Activity.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages.IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.

Copper ions trigger disassembly of neurokinin B functional amyloid and inhibit de novo assembly.

The formation of amyloid is considered an intrinsic ability of most polypeptides. It is a structure adopted by many neuropeptides and neurohormones during the formation of dense core vesicles in secretory cells, yet the mechanisms mediating assembly and disassembly of these amyloids remain unclear. Neurokinin B is a neuropeptide thought to form an amyloid in secretory cells. It is known to coordinate copper, but the physiological significance of metal binding is not known. In this work we explored the amyloid formation of neurokinin B and the impact that metals had on the aggregation behaviour. We show that the production of neurokinin B amyloid is dependent on the phosphate concentration, the pH and the presence of a histidine at position 3 in the primary sequence. Copper(II) and nickel(II) coordination to the peptide, which requires the histidine imidazole group, completely inhibits amyloid formation, whereas zinc(II) slows, but does not inhibit fibrillogenesis. Furthermore, we show that copper(II) can rapidly disassemble preformed neurokinin B amyloid. This work identifies a role for copper in neurokinin B structure and reveals a mechanism for amyloid assembly and disassembly dependent on metal coordination.

A gonadotropin-releasing hormone type neuropeptide with a high affinity binding site for copper(ii) and nickel(ii).

In vertebrates gonadotropin-releasing hormone I (GnRH-I) is a key regulator of reproductive development and function. The receptor-binding activity of human GnRH-I can be modified by the presence of divalent copper. Thus, copper binding to N-terminal amino acids in GnRH-I induces structural changes that influence receptor interactions and downstream intracellular signalling cascades. It is not known if copper-binding is restricted to human GnRH-I or if it is also a feature of GnRH-type peptides that have been identified in other taxa. To investigate this, we have characterised copper binding to a recently discovered GnRH-type peptide from the starfish Asterias rubens (ArGnRH). Using a range of spectroscopic and biophysical techniques we show that this peptide can bind copper(ii) and nickel(ii). Copper(ii) is bound in a square-planar, high-affinity (Kd ∼ 10-12 M) site incorporating four nitrogen donor atoms from a histidine imidazole group, two amides and the N-terminal amine group. The ArGnRH copper affinity and geometry are quite different to GnRH-I suggesting the copper sites have evolved to suit the environment the peptides are exposed to. By comparing the copper binding sites in ArGnRH and human GnRH-I and conducting a phylogenetic analysis of GnRH-type peptide sequences from a range of species, we predict that copper-binding is an evolutionarily ancient feature of GnRH-type peptides that has been retained, modified or lost in different lineages.

Endocytosis of G Protein-Coupled Receptors and Their Ligands: Is There a Role in Metal Trafficking?

The prevalence of metal dysregulation in many neurodegenerative and neurocognitive disorders has compelled many studying such diseases to investigate the mechanisms underlying metal regulation in the central nervous system. Metal homoeostasis is often complex, with sophisticated, multilayered pathways in operation. G protein-coupled receptors are omnipresent on cell membranes and have intriguing mechanisms of endocytosis and trafficking that may be useful in metal homoeostasis. Indeed, many receptors and/or their cognate ligands are able to bind metals, and in many cases metals are considered to have neuromodulatory roles as a result of receptor binding. In this mini-review, we outline the structural and functional aspects of G protein-coupled receptors with a focus on the mechanisms leading to endocytosis and cellular trafficking. We further highlight how this may help in the trafficking of metal ions, notably copper.

Neurokinin B and serum albumin limit copper binding to mammalian gonadotropin releasing hormone.

Gonadotropin releasing hormone (GnRH) triggers secretion of luteinizing hormone and follicle stimulating hormone from gonadotropic cells in the anterior pituitary gland. GnRH is able to bind copper, and both in vitro and in vivo studies have suggested that the copper-GnRH complex is more potent at triggering gonadotropin release than GnRH alone. However, it remains unclear whether copper-GnRH is the active species in vivo. To explore this we have estimated the GnRH-copper affinity and have examined whether GnRH remains copper-bound in the presence of serum albumin and the neuropeptide neurokinin B, both copper-binding proteins that GnRH will encounter in vivo. We show that GnRH has a copper dissociation constant of ∼0.9 × 10-9 M, however serum albumin and neurokinin B can extract metal from the copper-GnRH complex. It is therefore unlikely that a copper-GnRH complex will survive transit through the pituitary portal circulation and that any effect of copper must occur outside the bloodstream in the absence of neurokinin B.

RNA-Associated Early-Stage Antiviral Factor Is a Major Component of Lv2 Restriction.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication in human cells is restricted at early postentry steps by host inhibitory factors. We previously described and characterized an early-phase restriction of HIV-1 and -2 replication in human cell lines, primary macrophages, and peripheral blood mononuclear cells. The restriction was termed lentiviral restriction 2 (Lv2). The viral determinants of Lv2 susceptibility mapped to the HIV-2 envelope (Env) and capsid (CA). We subsequently reported a whole-genome small interfering RNA screening for factors involved in HIV that identified RNA-associated early-stage antiviral factor (REAF). Using HIV-2 chimeras of susceptible and nonsusceptible viruses, we show here that REAF is a major component of the previously described Lv2 restriction. Further studies of the viral CA demonstrate that the CA mutation I73V (previously called I207V), a potent determinant for HIV-2, is a weak determinant of susceptibility for HIV-1. More potent CA determinants for HIV-1 REAF restriction were identified at P38A, N74D, G89V, and G94D. These results firmly establish that in HIV-1, CA is a strong determinant of susceptibility to Lv2/REAF. Similar to HIV-2, HIV-1 Env can rescue sensitive CAs from restriction. We conclude that REAF is a major component of the previously described Lv2 restriction.IMPORTANCE Measures taken by the host cell to combat infection drive the evolution of pathogens to counteract or sidestep them. The study of such virus-host conflicts can point to possible weaknesses in the arsenal of viruses and may lead to the rational design of antiviral agents. Here we describe our discovery that the host restriction factor REAF fulfills the same criteria previously used to describe lentiviral restriction (Lv2). We show that, like the HIV-2 CA, the CA of HIV-1 is a strong determinant of Lv2/REAF susceptibility. We illustrate how HIV counteracts Lv2/REAF by using an envelope with alternative routes of entry into cells.

Nitroxide Spin-Labelling and Its Role in Elucidating Cuproprotein Structure and Function.

Copper is one of the most abundant biological metals, and its chemical properties mean that organisms need sophisticated and multilayer mechanisms in place to maintain homoeostasis and avoid deleterious effects. Studying copper proteins requires multiple techniques, but electron paramagnetic resonance (EPR) plays a key role in understanding Cu(II) sites in proteins. When spin-labels such as aminoxyl radicals (commonly referred to as nitroxides) are introduced, then EPR becomes a powerful technique to monitor not only the coordination environment, but also to obtain structural information that is often not readily available from other techniques. This information can contribute to explaining how cuproproteins fold and misfold. The theory and practice of EPR can be daunting to the non-expert; therefore, in this mini review, we explore how nitroxide spin-labelling can be used to help the inorganic biochemist gain greater understanding of cuproprotein structure and function in vitro and how EPR imaging may help improve understanding of copper homoeostasis in vivo.

Retroviral restriction: nature's own solution.

PURPOSE OF REVIEW: The present review will discuss recent advances in the development of anti-HIV therapies inspired by studies of the mechanisms of host restriction factor-mediated resistance to HIV infection. RECENT FINDINGS: Manipulating the interplay between host cell restriction factors and viral accessory factors that overcome them can potentially be therapeutically useful. Preliminarily successful therapies - some of which are entering clinical trials - either inhibit the ability of virus to evade restriction factor-mediated immunity, or promote intracellular levels of restriction factors. These aims are achieved by multiple means, which are discussed. SUMMARY: Many restriction factors appear to provide potentially useful targets for anti-HIV therapies, so time and interest should be invested in investigating ways to successfully therapeutically manipulate restriction factor-mediated immunity.

The antimicrobial efficacy and DNA binding activity of the copper(II) complexes of 3,4,7,8-tetramethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline and 1,2-diaminocyclohexane.

Four copper(II) complexes of the general structure [Cu(L1)(L2)]2+, where L1 is (1S,2S)-diaminocyclohexane or (1R,2R)-diaminocyclohexane and L2 is 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) or 4,7-diphenyl-1,10-phenanthroline (DIP), have been investigated in this study for their antimicrobial activity, short-term antimicrobial efficacy, and in vitro DNA-binding affinity. Against an expanded panel of bacterial and fungal strains in 12 species, minimal inhibitory concentrations (MIC) for these metallocomplexes were determined. The data confirmed our previous finding that they are effective against Gram-positive bacteria (MIC 5.6-13.1µM), with DIP coordinated complexes more so than TMP counterparts. Additionally, novel and significant findings were obtained here for these copper(II) complexes. While the four metallocomplexes exhibited high anti-Candida yeast activity (MIC 13.1-26.1µM), they demonstrated stronger anti-fungal activity against the drug-resistant Candida krusei (MIC 13.1µM and 22.6µM for TMP and DIP complexes, respectively) than the anti-fungal agent, 5-fluorocytosine. Fluorescence cell viability assays revealed that these complexes exert faster antibacterial effect than ampicillin as their inhibition against Staphylococcus aureus and Enterococcus faecalis were significantly evident within 0.5h of exposure compared to ampicillin. Similarly, these complexes but not ampicillin demonstrated bactericidal activity in non-proliferating conditions. All complexes exhibited DNA binding affinities similar to that of the known DNA intercalator, ethidium bromide (Ka ~105M-1) in linear dichroism binding studies and fluorescent dye displacement assays. Taken together, these findings imply that the four copper(II) complexes have different modes of action to the established antibiotics such as ampicillin and 5-fluorocytosine, and provide further insight into development of effective antimicrobial metallocomplexes.

Endocytosis of the tachykinin neuropeptide, neurokinin B, in astrocytes and its role in cellular copper uptake.

The tachykinin neuropeptide, neurokinin B (NKB), belongs to a family of peptides having diverse roles in the brain. NKB, along with several other tachykinins, has been identified as a copper-binding peptide, however the physiological relevance of the binding is unclear. Previously, NKB was shown to limit the ability of copper to enter astrocytes and disrupt calcium homeostasis and it was thought that the peptide was sequestering the metal extracellularly. Here we use a fluorescein-labelled NKB peptide (F-NKB) to show that NKB is not retained extracellularly, but is endocytosed within 10-20min after addition to the cell media. The endocytosis is not inhibited when NKB is delivered as a copper-complex, [CuII(F-NKB)2]. Endocytosis of NKB can increase intracellular copper. Comparison to cells cultured in copper-free buffer indicated that apo-NKB can facilitate uptake of copper found in normal culture media. To achieve this NKB must compete with a variety of copper proteins, and we show that NKB can successfully compete with copper-binding peptides derived from the prion protein, itself associated with Cu(II) and Zn(II) metabolism. We suggest a mechanism of receptor mediated endocytosis to account for the observations.

Identification of a neuropeptide precursor protein that gives rise to a "cocktail" of peptides that bind Cu(II) and generate metal-linked dimers.

BACKGROUND: Neuropeptides with an Amino Terminal Cu(II), Ni(II) Binding (ATCUN) motif (H2N-xxH) bind Cu(II)/Ni(II) ions. Here we report the novel discovery of a neuropeptide precursor that gives rise to a "cocktail" of peptides that bind Cu(II)/Ni(II) and form ternary complexes--the L-type SALMFamide precursor in the starfish Asterias rubens. METHODS: Echinoderm transcriptome sequence data were analysed to identify transcripts encoding precursors of SALMFamide-type neuropeptides. The sequence of the L-type SALMFamide precursor in the starfish Asterias rubens was confirmed by cDNA sequencing and peptides derived from this precursor (e.g. AYHSALPF-NH2, GYHSGLPF-NH2 and LHSALPF-NH2) were synthesized. The ability of these peptides to bind metals was investigated using UV/Vis, NMR, circular dichroism and EPR spectroscopy. RESULTS: AYHSALPF-NH2 and GYHSGLPF-NH2 bind Cu(II) and Ni(II) and generate metal-linked dimers to form ternary complexes with LHSALPF-NH2. Investigation of the evolutionary history of the histidine residue that confers these properties revealed that it can be traced to the common ancestor of echinoderms, which is estimated to have lived ~500 million years ago. However, L-type precursors comprising multiple SALMFamides with the histidine residue forming an ATCUN motif appears to be a feature that has evolved uniquely in starfish (Asteroidea). GENERAL SIGNIFICANCE: The discovery of a SALMFamide-type neuropeptide precursor protein that gives rise to a "cocktail" of peptides that bind metal ions and generate metal-linked dimers provides a new insight on ATCUN motif-containing neuropeptides. This property of L-type SALMFamides in the Asteroidea may be associated with a role in regulation of the unusual extra-oral feeding behaviour of starfish.

Bioactivity and structural properties of chimeric analogs of the starfish SALMFamide neuropeptides S1 and S2.

The starfish SALMFamide neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide) are the prototypical members of a family of neuropeptides that act as muscle relaxants in echinoderms. Comparison of the bioactivity of S1 and S2 as muscle relaxants has revealed that S2 is ten times more potent than S1. Here we investigated a structural basis for this difference in potency by comparing the bioactivity and solution conformations (using NMR and CD spectroscopy) of S1 and S2 with three chimeric analogs of these peptides. A peptide comprising S1 with the addition of S2's N-terminal tetrapeptide (Long S1 or LS1; SGPYGFNSALMFamide) was not significantly different to S1 in its bioactivity and did not exhibit concentration-dependent structuring seen with S2. An analog of S1 with its penultimate residue substituted from S2 (S1(T); GFNSALTFamide) exhibited S1-like bioactivity and structure. However, an analog of S2 with its penultimate residue substituted from S1 (S2(M); SGPYSFNSGLMFamide) exhibited loss of S2-type bioactivity and structural properties. Collectively, our data indicate that the C-terminal regions of S1 and S2 are the key determinants of their differing bioactivity. However, the N-terminal region of S2 may influence its bioactivity by conferring structural stability in solution. Thus, analysis of chimeric SALMFamides has revealed how neuropeptide bioactivity is determined by a complex interplay of sequence and conformation.