A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.

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Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time-and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against S. aureus at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin, S. aureus strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by S. aureus contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.

Fragile X mental retardation protein, FMRP, is absent in patients with fragile X syndrome, a common form of mental retardation. FMRP is a nucleocytoplasmic RNA binding protein that is primarily associated with polyribosomes. FMRP is believed to be a translational repressor and may regulate the translation of certain mRNAs at the base of dendritic spines in neurons. However, little is known about the regulation of FMRP. Using mass spectrometry and site-directed mutagenesis, we show that FMRP is phosphorylated between residues 483 and 521, N-terminal to the RGG box, both in murine brain and in cultured cells. Primary phosphorylation occurs on the highly conserved serine 499, which triggers hierarchical phosphorylation of nearby serines. FMRP is phosphorylated within 2-4 h of synthesis, however, phosphorylation has no effect on the half-life of the protein. In contrast to the Drosophila ortholog dFxr, the phosphorylation status of mammalian FMRP does not influence its association with specific mRNAs in vivo. However, we find unphosphorylated FMRP associated with actively translating polyribosomes while a fraction of phosphorylated FMRP is associated with apparently stalled polyribosomes. Our data suggest that the phosphorylation may regulate FMRP and that the release of FMRP-induced translational suppression may involve a dephosphorylation signal.

14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PH-CVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys 49 , Arg 56 , Arg 60 , and Arg 127 ), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 Å apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.