Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.

Active barnase variants with completely random hydrophobic cores.

The central structural feature of natural proteins is a tightly packed and highly ordered hydrophobic core. If some measure of exquisite, native-like core packing is necessary for enzymatic function, this would constitute a significant obstacle to the development of novel enzymes, either by design or by natural or experimental evolution. To test the minimum requirements for a core to provide sufficient structural integrity for enzymatic activity, we have produced mutants of the ribonuclease barnase in which 12 of the 13 core residues have together been randomly replaced by hydrophobic alternatives. Using a sensitive biological screen, we find that a strikingly high proportion of these mutants (23%) retain enzymatic activity in vivo. Further substitution at the 13th core position shows that a similar proportion of completely random hydrophobic cores supports enzyme function. Of the active mutants produced, several have no wild-type core residues. These results imply that hydrophobicity is nearly a sufficient criterion for the construction of a functional core and, in conjunction with previous studies, that refinement of a crudely functional core entails more stringent sequence constraints than does the initial attainment of crude core function. Since attainment of crude function is the critical initial step in evolutionary innovation, the relatively scant requirements contributed by the hydrophobic core would greatly reduce the initial hurdle on the evolutionary pathway to novel enzymes. Similarly, experimental development of novel functional proteins might be simplified by limiting core design to mere specification of hydrophobicity and using iterative mutation-selection to optimize core structure.

The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters

Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat49–57 (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat49–57 were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat49–57 play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat49–57 in cellular uptake. In contrast, a 9-mer of l-arginine (R9) was 20-fold more efficient than Tat49–57 at cellular uptake as determined by Michaelis–Menton kinetic analysis. The d-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat49–57 play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat49–57 and even to r9. Overall, a transporter has been developed that is superior to Tat49–57, protease resistent, and more readily and economically prepared.