Affinity gradients drive copper to cellular destinations.

Copper is an essential trace element for eukaryotes and most prokaryotes. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we determine, through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymatic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodynamic data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein-protein interactions and therefore protein-protein specific recognition. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodynamic basis for the kinetic processes that lead to the distribution of cellular copper.

Solid-State NMR of PEGylated Proteins.

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.

Beta Amyloid Hallmarks: From Intrinsically Disordered Proteins to Alzheimer's Disease.

Beta amyloid protein (Aß) is one of the intrinsically disordered proteins associated with neurodegenerative diseases like Parkinson's, prion disease and Alzheimer's disease (AD) in particular. Although the direct involvement of Aß peptides in AD is well documented and their aggregative ability is closely related to their neurotoxicity, the precise mechanism of the neurotoxic effects of Aß peptides remains unclear. There is still a significant gap between the site-specific structural information and the complex structural diversity of Aß amyloids. The description of the structural polymorphisms of Aß amyloids can provide valuable information of the molecular basis of AD onset-progress and is essential for comprehension of the Aß aggregation pathways, in particular its structural evolution. In this review we tried to illustrate the emerging trend of defining several human neurodegenerative disorders as syndromes of protein folding and oligomerization through the example of AD.

Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks.

The growing recognition of the several roles that intrinsically disordered proteins play in biology places an increasing importance on protein sample availability to allow the characterization of their structural and dynamic properties. The sample preparation is therefore the limiting step to allow any biophysical method being able to characterize the properties of an intrinsically disordered protein and to clarify the links between these properties and the associated biological functions. An increasing array of tools has been recruited to help prepare and characterize the structural and dynamic properties of disordered proteins. This chapter describes their sample preparation, covering the most common drawbacks/barriers usually found working in the laboratory bench. We want this chapter to be the bedside book of any scientist interested in preparing intrinsically disordered protein samples for further biophysical analysis.

Human superoxide dismutase 1 (hSOD1) maturation through interaction with human copper chaperone for SOD1 (hCCS).

Copper chaperone for superoxide dismutase 1 (SOD1), CCS, is the physiological partner for the complex mechanism of SOD1 maturation. We report an in vitro model for human CCS-dependent SOD1 maturation based on the study of the interactions of human SOD1 (hSOD1) with full-length WT human CCS (hCCS), as well as with hCCS mutants and various truncated constructs comprising one or two of the protein's three domains. The synergy between electrospray ionization mass spectrometry (ESI-MS) and NMR is fully exploited. This is an in vitro study of this process at the molecular level. Domain 1 of hCCS is necessary to load hSOD1 with Cu(I), requiring the heterodimeric complex formation with hSOD1 fostered by the interaction with domain 2. Domain 3 is responsible for the catalytic formation of the hSOD1 Cys-57-Cys-146 disulfide bond, which involves both hCCS Cys-244 and Cys-246 via disulfide transfer.

Mechanistic aspects of hSOD1 maturation from the solution structure of Cu(I) -loaded hCCS domain 1 and analysis of disulfide-free hSOD1 mutants.

Superoxide dismutase 1 (SOD1) maturation within the cell is mainly accomplished with the SOD1-specific chaperone, CCS, a dimeric protein with three distinct domains in each monomer. We recently showed that the first domain of human CCS (hCCSD1) is responsible for copper transfer to its protein partner, human SOD1 (hSOD1). The NMR solution structure of the copper(I)-loaded form of hCCSD1 reported here contributes further to characterization of the copper-transfer mechanism to hSOD1. NMR spectroscopy was also used to examine the hSOD1 mutants C57A, C146A, and C57A/C146A, which are unable to form the structurally conserved disulfide bond in SOD1, in order to investigate the role of these cysteines during hSOD1 copper acquisition. Together, the information on both hCCS and hSOD1, along with a sequence analysis of eukaryotic CCSD1, allows us to propose important mechanistic aspects regarding the copper-transfer process from hCCS to hSOD1.

Sco proteins are involved in electron transfer processes.

Sco proteins are widespread in eukaryotic and in many prokaryotic organisms. They have a thioredoxin-like fold and bind a single copper(I) or copper(II) ion through a CXXXC motif and a conserved His ligand, with both tight and weak affinities. They have been implicated in the assembly of the Cu(A) site of cytochrome c oxidase as copper chaperones and/or thioredoxins. In this work we have structurally characterized a Sco domain which is naturally fused with a typical electron transfer molecule, i.e., cytochrome c, in Pseudomonas putida. The thioredoxin-like Sco domain does not bind copper(II), binds copper(I) with weak affinity without involving the conserved His, and has redox properties consisting of a thioredoxin activity and of the ability of reducing copper(II) to copper(I), and iron(III) to iron(II) of the cytochrome c domain. These findings indicate that the His ligand coordination is the discriminating factor for introducing a metallochaperone function in a thioredoxin-like fold, typically responsible for electron transfer processes. A comparative structural analysis of the Sco domain from P. putida versus eukaryotic Sco proteins revealed structural determinants affecting the formation of a tight-affinity versus a weak-affinity copper binding site in Sco proteins.

A multifunctional organosilica cross-linker for the bio-conjugation of gold nanorods.

We report on the use of organosilica shells to couple gold nanorods to functional peptides and modulate their physiochemical and biological profiles. In particular, we focus on the case of cell penetrating peptides, which are used to load tumor-tropic macrophages and implement an innovative drug delivery system for photothermal and photoacoustic applications. The presence of organosilica exerts subtle effects on multiple parameters of the particles, including their size, shape, electrokinetic potential, photostability, kinetics of endocytic uptake and cytotoxicity, which are investigated by the interplay of colorimetric methods and digital holographic microscopy. As a rule of thumb, as the thickness of organosilica increases from none to ∼30nm, we find an improvement of the photophysical performances at the expense of a deterioration of the biological parameters. Therefore, detailed engineering of the particles for a certain application will require a careful trade-off between photophysical and biological specifications.

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants.

Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone.