Skin rejuvenation using cosmetic products containing growth factors, cytokines, and matrikines: a review of the literature.

Skin aging is primarily due to alterations in the dermal extracellular matrix, especially a decrease in collagen I content, fragmentation of collagen fibrils, and accumulation of amorphous elastin material, also known as elastosis. Growth factors and cytokines are included in several cosmetic products intended for skin rejuvenation because of their ability to promote collagen synthesis. Matrikines and matrikine-like peptides offer the advantage of growth factor-like activities but better skin penetration due to their much smaller molecular size. In this review, we summarize the commercially available products containing growth factors, cytokines, and matrikines for which there is evidence that they promote skin rejuvenation.

Kinetic consequences of introducing a proximal selenocysteine ligand into cytochrome P450cam.

The structural, electronic, and catalytic properties of cytochrome P450cam are subtly altered when the cysteine that coordinates to the heme iron is replaced with a selenocysteine. To map the effects of the sulfur-to-selenium substitution on the individual steps of the catalytic cycle, we conducted a comparative kinetic analysis of the selenoenzyme and its cysteine counterpart. Our results show that the more electron-donating selenolate ligand has only negligible effects on substrate, product, and oxygen binding, electron transfer, catalytic turnover, and coupling efficiency. Off-pathway reduction of oxygen to give superoxide is the only step significantly affected by the mutation. Incorporation of selenium accelerates this uncoupling reaction approximately 50-fold compared to sulfur, but because the second electron transfer step is much faster, the impact on overall catalytic turnover is minimal. Density functional theory calculations with pure and hybrid functionals suggest that superoxide formation is governed by a delicate interplay of spin distribution, spin state, and structural effects. In light of the remarkably similar electronic structures and energies calculated for the sulfur- and selenium-containing enzymes, the ability of the heavier atom to enhance the rate of spin crossover may account for the experimental observations. Because the selenoenzyme closely mimics wild-type P450cam, even at the level of individual steps in the reaction cycle, selenium represents a unique mechanistic probe for analyzing the role of the proximal ligand and spin crossovers in P450 chemistry.

C-terminal domain of archaeal O-phosphoseryl-tRNA kinase displays large-scale motion to bind the 7-bp D-stem of archaeal tRNA(Sec).

O-Phosphoseryl-tRNA kinase (PSTK) is the key enzyme in recruiting selenocysteine (Sec) to the genetic code of archaea and eukaryotes. The enzyme phosphorylates Ser-tRNA(Sec) to produce O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) that is then converted to Sec-tRNA(Sec) by Sep-tRNA:Sec-tRNA synthase. Earlier we reported the structure of the Methanocaldococcus jannaschii PSTK (MjPSTK) complexed with AMPPNP. This study presents the crystal structure (at 2.4-Å resolution) of MjPSTK complexed with an anticodon-stem/loop truncated tRNA(Sec) (Mj*tRNA(Sec)), a good enzyme substrate. Mj*tRNA(Sec) is bound between the enzyme's C-terminal domain (CTD) and N-terminal kinase domain (NTD) that are connected by a flexible 11 amino acid linker. Upon Mj*tRNA(Sec) recognition the CTD undergoes a 62-Å movement to allow proper binding of the 7-bp D-stem. This large reorganization of the PSTK quaternary structure likely provides a means by which the unique tRNA(Sec) species can be accurately recognized with high affinity by the translation machinery. However, while the NTD recognizes the tRNA acceptor helix, shortened versions of MjPSTK (representing only 60% of the original size, in which the entire CTD, linker loop and an adjacent NTD helix are missing) are still active in vivo and in vitro, albeit with reduced activity compared to the full-length enzyme.

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.

The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.

Closely related antibody receptors exploit fundamentally different strategies for steroid recognition.

Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu(H47)Trp and Arg(H100)Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 Leu(H47)Trp/Arg(H100)Trp double mutant (1E9dm) as an unliganded Fab at 2.05 A resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 A. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.

Switching antibody specificity through minimal mutation.

Antibody 1E9, which was elicited with a hexachloronorbornene derivative and catalyzes the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide with high efficiency, was successfully reengineered to bind a range of structurally diverse steroids with nanomolar affinities. Remarkably, two mutations (Leu(H47)Trp/Arg(H100)Trp) out of 36 total sequence differences suffice to switch the selectivity of 1E9 to that of the progesterone-binding antibody DB3. In contrast to the double mutant, which tightly binds multiple steroids with differently configured A-B ring junctions, the individual Leu(H47)Trp and Arg(H100)Trp single mutants both exhibit significantly greater specificity than DB3, preferentially binding 5alpha-pregnan-3beta-ol-20-one (K(d) approximately 5 nM) over other steroids. These findings illustrate how easily differently shaped binding pockets can be created through subtle changes to the same primordial germ line template.