Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

An Electrochemical Biosensor Architecture Based on Protein Folding Supports Direct Real-Time Measurements in Whole Blood.

The ability to monitor drug and biomarker concentrations in the body with high frequency and in real time would revolutionize our understanding of biology and our capacity to personalize medicine. The few in vivo molecular sensors that currently exist, however, all rely on the specific chemical or enzymatic reactivity of their targets and thus are not generalizable. In response, we demonstrate here an electrochemical sensing architecture based on binding-induced protein folding that is 1) independent of the reactivity of its targets, 2) reagentless, real-time, and with a resolution of seconds, and 3) selective enough to deploy in undiluted bodily fluids. As a proof of principle, we use the SH3 domain from human Fyn kinase to build a sensor that discriminates between the protein's peptide targets and responds rapidly and quantitatively even when challenged in whole blood. The resulting sensor architecture could drastically expand the chemical space accessible to continuous, real-time biosensors.