A peptide self-assembled monolayer (SAM) was designed to bind His-tagged biomolecules for surface plasmon resonance (SPR) bioanalysis, which was applied for the determination of K(d) for small ligand screening against CD36. Nonspecific adsorption could be minimized using penta- and hexa-peptide monolayers. In particular, monolayers consisting of 3-mercaptopropionyl-leucinyl-histidinyl-aspartyl-leucinyl-histidinyl-aspartic acid (3-Mpa-LHDLHD) exhibited little (12 ng cm(-2)) nonspecific adsorption in crude serum. Modification of this peptide monolayer with Nα,Nα-bis(carboxymethyl)-L-lysine gave a surface competent for binding His-tagged proteins, as demonstrated using enzyme (human dihydrofolate reductase), protein/antibody and receptor (CD36) examples. Immobilization featured chelation of copper and the His-tagged protein by the peptide monolayer, which could be recycled by removing the copper using imidazole washes prior to reuse.

The telomerase holoenzyme responsible for maintaining telomeres in vertebrates requires many components in vivo , including dyskerin. Dyskerin binds and regulates the accumulation of the human telomerase RNA, hTR, as well as other non-coding RNAs that share the conserved H/ACA box motif. The precise mechanism by which dyskerin controls hTR levels is unknown, but is evidenced by defective hTR accumulation caused by substitutions in dyskerin, that are observed in the X-linked telomere biology disorder dyskeratosis congenita (X-DC). To understand the role of dyskerin in hTR accumulation, we analyzed X-DC substitutions K39E and K43E in the poorly characterized dyskerin N-terminus, and A353V within the canonical RNA binding domain (the PUA). These variants exhibited impaired binding to hTR and polyadenylated hTR species, while interactions with other H/ACA RNAs appear largely unperturbed by the N-terminal substitutions. hTR accumulation and telomerase activity defects of dyskerin-deficient cells were rescued by wildtype dyskerin but not the variants. hTR 3′ extended or polyadenylated species did not accumulate, suggesting hTR precursor degradation occurs upstream of mature complex assembly in the absence of dyskerin binding. Our findings demonstrate that the dyskerin-hTR interaction mediated by PUA and N-terminal residues of dyskerin is crucial to prevent unchecked hTR degradation.

The nuclear and subnuclear compartmentalization of the telomerase-associated protein and H/ACA ribonucleoprotein component dyskerin is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein.