Department of Biological Chemistry, UCLA, David Geffen School of Medicine

Multiple experiments using diabetes animal models have demonstrated the antihyperglycemic properties of several Mexican Copalchi species, mainly Hintonia latiflora (HLA) and Hintonia standleyana (HSL). The active compounds of these plants have recently been characterized and their therapeutic mechanisms proposed. Though widely studied in animals, evidence indicating therapeutic efficacy in patients with non-insulin-dependent diabetes mellitus (NIDDM) is limited. Still, copalchis have been utilized as a folk remedy, and in Germany as a marketed drug, for managing NIDDM. The risks and benefits of using this drug warrant critical analysis. This review summarizes research evidence for the antidiabetic efficacy of Copalchis in NIDDM animal models, recent toxicity findings, and limited clinical findings in human subjects.

Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser¹²⁹ (pS¹²⁹-α-syn), whereas >90% pS¹²⁹-α-syn may be found in LBs, suggesting that pS¹²⁹-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS¹²⁹-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)'s electrochemiluminescence platform to create a new assay for sensitive detection of pS¹²⁹-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS¹²⁹-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 10⁴ pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS¹²⁹-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS¹²⁹-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.

Knowing the genes involved in quantitative traits provides an entry point to understanding the biological bases of behavior, but there are very few examples where the pathway from genetic locus to behavioral change is known. To explore the role of specific genes in fear behavior, we mapped three fear-related traits, tested fourteen genes at six quantitative trait loci (QTLs) by quantitative complementation, and identified six genes. Four genes, Lamp, Ptprd, Nptx2, and Sh3gl, have known roles in synapse function; the fifth, Psip1, was not previously implicated in behavior; and the sixth is a long non-coding RNA, 4933413L06Rik, of unknown function. Variation in transcriptome and epigenetic modalities occurred preferentially in excitatory neurons, suggesting that genetic variation is more permissible in excitatory than inhibitory neuronal circuits. Our results relieve a bottleneck in using genetic mapping of QTLs to uncover biology underlying behavior and prompt a reconsideration of expected relationships between genetic and functional variation.

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (^CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created ^CdSrtA^3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. ^CdSrtA^3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that ^CdSrtA^3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.