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.

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.

Mitochondria are able to modulate cell state and fate during normal and pathophysiologic conditions through a nuclear-mediated mechanism collectively termed as a retrograde response. Our previous studies in Drosophila melanogaster have clearly established that progress through the cell cycle is precisely regulated by the intrinsic activity of the mitochondrion by specific signaling cascades mounted by the cell. As a means to further our understanding of how mitochondrial energy status affects nuclear control of basic cell decisions, we have employed Affymetrix microarray-based transcriptional profiling of Drosophila S2 cells knocked down for the gene encoding subunit Va of the complex IV of the mitochondrial electron transport chain. The profiling data identify transcriptional upregulation of glycolytic genes, and metabolic studies confirm this increase in glycolysis. The data provide a model of the shift of metabolism from a predominately oxidative state toward a predominately aerobic glycolytic state mediated through transcriptional control. The transcriptional changes alter many signaling systems, including p53, insulin, hypoxia-induced factor α, and conserved mitochondrial retrograde responses. This rich dataset provides many novel targets for further understanding the mechanism whereby the mitochondrion manages energy substrate disposition and directs cellular fate decisions.