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.

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.

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.

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.