Colin S. Burns

Copper Coordination in the Full-Length, Recombinant Prion Protein

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991-4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760-13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.

Peculiarities of Copper Binding to α-Synuclein

Abstract Heavy metals have been implicated as the causative agents for the pathogenesis of the most prevalent neurodegenerative disease. Various mechanisms have been proposed to explain the toxic effects of metals ranging from metal-induced oxidation of protein to metal-induced changes in the protein conformation. Aggregation of α-synuclein is implicated in Parkinsons disease (PD), and various metals, including copper, constitute a prominent group of α-synuclein aggregation enhancers. In this study, we have systematically characterized the α-synuclein-Cu2+ binding sites and analyzed the possible role of metal binding in α-synuclein fibrillation using a set of biophysical techniques, such as electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), circular dichroism (CD), and size exclusion chromatography (SEC). Our analyses indicated that α-synuclein possesses at least two binding sites for Cu2+. We have been able to locate one of the binding sites in the N-terminal region. Furthermore, based on the EPR studies of model peptides and β-synuclein, we concluded that the suspected His residue did not appear to participate in strong Cu2+ binding.

Influence of Prothymosin-α on HIV-1 Target Cells

Abstract:  The important role of CD8+ T cells in controlling HIV‐1 infection through the innate as well as the adaptive immune system is well established. In addition to the major histocompatibility complex (MHC)‐dependent cytotoxic activity of CD8+ T cells, they produce soluble factors that suppress HIV‐1 replication in an MHC‐independent manner. Several of those factors have been identified, including β‐chemokines, Rantes, MIP‐1α, MIP‐1β, and MDC. We previously identified that prothymosin alpha (ProTα) in the conditioned medium of HVS transformed CD8+ T cells was a potent inhibitor of HIV‐1 replication following proviral integration. In this report we further characterize the anti‐HIV‐1 activity of ProTα by demonstrating its target‐cell specificity, distinction from additional inhibitors of HIV‐1 transcription in CD8+ T cell supernatants, as well as the differential regulation of host cell antiviral genes that could impact HIV‐1 replication. These genes include a number of transcription factors as well IFN‐α‐inducible genes including PKR, IRF1, and Rantes, in the absence of induction of IFN‐α. These data suggest that the anti‐HIV‐1 activity of ProTα is mediated through the modulation of a number of genes that have been reported to suppress HIV‐1 replication including the dysregulation of transcription factors and the induction of PKR and Rantes mRNA.

Zinc Deposition During ESI-MS Analysis of Peptide-Zinc Complexes

Electrospray ionization (ESI) mass spectrometry (MS) has proven to be an extremely powerful technique for studying the stoichiometry and binding strength of peptide-metal complexes. We have found a significant new problem in the ESI-MS of zinc-peptide systems involving the deposition of zinc in the ESI emitter. This deposition of zinc during the experiment removes a significant amount of zinc ions from the solution, impacting the resulting mass spectral intensities used to quantify the amount of the zinc-bound species. Analysis of infused zinc-peptide samples with atomic absorption spectrometry and with a custom-built nanoflow ESI source confirms the alteration of the analyte solutions with positive or negative or no potential applied to the emitter. Ultimately, the location of the zinc deposition was determined to be the stainless steel emitter. The use of a custom-built nanoESI interface using glass emitters was found to mitigate the zinc deposition problem. The phenomenon of metal deposition warrants further investigation as it may not be limited to just zinc and may represent a significant obstacle in the ESI-MS analysis of all protein-metal systems.

Calorimetric investigation of copper binding in the N-terminal region of the prion protein at low copper loading: evidence for an entropically favorable first binding event.

Although the Cu2+-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu2+, the Cu2+-loading mechanism is just beginning to come into view. Because the Cu2+-binding modes at low and intermediate Cu2+ occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper–prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.g., ΔH and ΔS) have been measured, thus leaving undetermined the enthalpic and entropic factors that govern the free energy of Cu2+ binding to the prion protein. In this study, isothermal titration calorimetry (ITC) was used to quantify the thermodynamic parameters (K, ΔG, ΔH, and TΔS) of Cu2+ binding to a peptide, PrP(23–28, 57–98), that encompasses the majority of the residues implicated in Cu2+ binding by full-length PrP. Use of the buffer N-(2-acetomido)-aminoethanesulfonic acid (ACES), which is also a well-characterized Cu2+ chelator, allowed for the isolation of the two highest affinity binding events. Circular dichroism spectroscopy was used to characterize the different binding modes as a function of added Cu2+. The Kd values determined by ITC, 7 and 380 nM, are well in line with those reported by others. The first binding event benefits significantly from a positive entropy, whereas the second binding event is enthalpically driven. The thermodynamic values associated with Cu2+ binding by the Aβ peptide, which is implicated in Alzheimer’s disease, bear striking parallels to those found here for the prion protein.

Synthesis and Evaluation of the Novel Prostamide, 15-Deoxy, Δ12,14-Prostamide J2, as a Selective Antitumor Therapeutic

15-deoxy, Δ12,14-prostaglandin J2-ethanolamide, also known as 15-deoxy, Δ12,14-prostamide J2 (15d-PMJ2) is a novel product of the metabolism of arachidonoyl ethanolamide (AEA) by COX-2. 15d-PMJ2 preferentially induced cell death and apoptosis in tumorigenic A431 keratinocytes and B16F10 melanoma cells compared with nontumorigenic HaCaT keratinocytes and Melan-A melanocytes. Activation of the ER stress execution proteins, PERK and CHOP10, was evaluated to determine whether this process was involved in 15d-PMJ2 cell death. 15d-PMJ2 increased the phosphorylation of PERK and expression of CHOP10 in tumorigenic but not nontumorigenic cells. The known ER stress inhibitors, salubrinal and 4-phenylbutaric acid, significantly inhibited 15d-PMJ2–mediated apoptosis, suggesting ER stress as a primary apoptotic mediator. Furthermore, the reactive double bond present within the cyclopentenone structure of 15d-PMJ2 was identified as a required moiety for the induction of ER stress apoptosis. The effect of 15d-PMJ2 on B16F10 melanoma growth was also evaluated by dosing C57BL/6 mice with 0.5 mg/kg 15d-PMJ2. Tumors of animals treated with 15d-PMJ2 exhibited significantly reduced growth and mean weights compared with vehicle and untreated animals. TUNEL and IHC analysis of tumor tissues showed significant cell death and ER stress in tumors of 15d-PMJ2–treated compared with control group animals. Taken together, these findings suggest that the novel prostamide, 15d-PMJ2, possesses potent antitumor activity in vitro and in vivo. Mol Cancer Ther; 16(5); 838–49. ©2017 AACR.

Field- and Temperature-Dependent 13C NMR Studies of the EDTA–Zn2+ Complex: Insight into Structure and Dynamics via Relaxation Measurements

The relaxation rates for the three different carbon types in EDTA (carbonyl, CH2 central, and CH2 lateral) were measured with and without Zn(2+) as a function of field strength and temperature. The use of different field strengths in combination with NOE measurements allowed for the contribution of each relaxation mechanism (chemical shift anisotropy; spin rotation; dipole-dipole) to the total relaxation rate for each carbon to be determined. Temperature studies allowed for determination of the activation energy (Ea) for the motions of each carbon type. The most surprising result was the observation that the τ(c) decreases significantly for the lateral carbon upon addition of Zn(2+) at neutral pH, going from 54 to 8.6 ps at 298 K. This appears to be a pH-dependent phenomenon as other reports indicate that τ(c) increases for the lateral carbon upon addition of Zn(2+) under strongly basic conditions.

The 4-pyridylmethyl ester as a protecting group for glutamic and aspartic acids: ‘flipping’ peptide charge states for characterization by positive ion mode ESI-MS

Use of the 4‐pyridylmethyl ester group for side‐chain protection of glutamic acid residues in solid‐phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI‐MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4‐pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI‐MS. Copyright

Zn2+-binding in the glutamate-rich region of the intrinsically disordered protein prothymosin-alpha

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein’s function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein’s ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.