A novel mechanism of lysosomal acid sphingomyelinase maturation: requirement for carboxyl-terminal proteolytic processing

Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn(2+))-independent lysosomal aSMase (L-SMase) and a Zn(2+)-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn(2+)-activated proenzyme to a mature 65 kDa, Zn(2+)-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn(2+)-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same M(r) as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function.     

In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS

High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r² = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with ²H₂O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of ²H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols.     

Adrenomedullary Secretion of DOPA, Catecholamines, Catechol Metabolites, and Neuropeptides

Abstract: Catecholamines and their metabolites have been proposed as markers of sympathetic nervous system stimulation. However, the adrenal medulla is a rich source of catecholamines and catecholamine metabolites and may play a significant role in plasma levels of these compounds. In addition to adrenal catecholamine metabolite efflux, the role of the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has not been fully evaluated. The simultaneous effluxes of catecholamines, metabolites, DOPA, and neuropeptides were measured in perfusates from isolated dog adrenals. The relative abundance of compounds detected consistently during unstimulated conditions was epinephrine ≫ norepinephrine > 3,4-dihydroxyphenylglycol > metanephrine > normetanephrine > dopamine > 3,4-dihydroxyphenylacetic acid > 3-methoxy-4-hydroxyphenylglycol ≥ DOPA ≫ [Met]enkephalin ≫ neuropeptide Y. Effluxes of analytes were not affected by cocaine and the ratios of catecholamines to metabolites increased dramatically with carbachol stimulation, consistent with negligible reuptake into adrenal cells. Thus, most of the 3,4-dihydroxyphenylglycol is expected to be derived from epinephrine and norepinephrine subsequent to translocation from chromaffin vesicles into the cytosol. The efflux of DOPA increased dramatically during stimulation with 30 µ M carbachol in a calcium-dependent manner. Efflux of DOPA during the initial stabilization period of the perfusion preparation declined exponentially, in parallel with the effluxes of the catecholamines and neuropeptides but not with metabolites. Evoked release of DOPA was Ca²⁺-dependent. These data suggest that DOPA can be stored and released exocytotically from chromaffin granules.     

Quantifying cholesterol synthesis in vivo using (2)H(2)O: enabling back-to-back studies in the same subject

The advantages of using (2)H(2)O to quantify cholesterol synthesis include i) homogeneous precursor labeling, ii) incorporation of (2)H via multiple pathways, and iii) the ability to perform long-term studies in free-living subjects. However, there are two concerns. First, the t(1/2) of tracer in body water presents a challenge when there is a need to acutely replicate measurements in the same subject. Second, assumptions are made regarding the number of hydrogens (n) that are incorporated during de novo synthesis. Our primary objective was to determine whether a step-based approach could be used to repeatedly study cholesterol synthesis a subject. We observed comparable changes in the (2)H-labeling of plasma water and total plasma cholesterol in African-Green monkeys that received five oral doses of (2)H(2)O, each dose separated by one week. Similar rates of cholesterol synthesis were estimated when comparing data in the group over the different weeks, but better reproducibility was observed when comparing replicate determinations of cholesterol synthesis in the same nonhuman primate during the respective dosing periods. Our secondary objective was to determine whether n depends on nutritional status in vivo; we observed n of ～25 and ～27 in mice fed a high-carbohydrate (HC) versus carbohydrate-free (CF) diet, respectively. We conclude that it is possible to acutely repeat studies of cholesterol synthesis using (2)H(2)O and that n is relatively constant.     

SAVVY? (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind,Randomized, Placebo-Controlled Trial in Ghana

Objective

The objective of this trial was to determine the effectiveness of 1.0% C31G (SAVVY) in preventing male-to-female vaginal transmission of HIV infection among women at high risk.

Methodology/Principal Findings

This was a Phase 3, double-blind, randomized, placebo-controlled trial. Participants made up to 12 monthly visits for HIV testing, adverse event reporting, and study product supply. The study was conducted between March 2004 and February 2006 in Accra and Kumasi, Ghana. We enrolled 2142 HIV-negative women at high risk of HIV infection, and randomized them to SAVVY or placebo gel. Main outcome measures were the incidence of HIV-1 and HIV-2 infection as determined by detection of HIV antibodies from oral mucosal transudate specimens and adverse events. We accrued 790 person-years of follow-up in the SAVVY group and 772 person-years in the placebo group. No clinically significant differences in the overall frequency of adverse events, abnormal pelvic examination findings, or abnormal laboratory results were seen between treatment groups. However, more participants in the SAVVY group reported reproductive tract adverse events than in the placebo group (13.0% versus 9.4%). Seventeen HIV seroconversions occurred; eight in participants randomized to SAVVY and nine in participants receiving placebo. The Kaplan-Meier estimates of the cumulative probability of HIV infection through 12 months were 0.010 in the SAVVY group and 0.011 in the placebo group (p = 0.731), with a hazard ratio (SAVVY versus placebo) of 0.88 (95% confidence interval 0.33, 2.27). Because of a lower-than-expected HIV incidence, we were unable to achieve the required number of HIV infections (66) to obtain the desired study power.

Conclusions/Significance

SAVVY was not associated with increased adverse events overall, but was associated with higher reporting of reproductive adverse events. Our data are insufficient to conclude whether SAVVY is effective at preventing HIV infection relative to placebo.

Trial Registration

ClinicalTrials.gov {\"type\":\"clinical-trial\",\"attrs\":{\"text\":\"NCT00129532\",\"term_id\":\"NCT00129532\"}}NCT00129532     

Measuring H(2)(18)O tracer incorporation on a QQQ-MS platform provides a rapid, transferable screening tool for relative protein synthesis

Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H(2)(18)O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H(2)(18)O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment.     

The human carbon budget: an estimate of the spatial distribution of metabolic carbon consumption and release in the United States

Carbon dioxide is taken up by agricultural crops and released soon after during the consumption of agricultural commodities. The global net impact of this process on carbon flux to the atmosphere is negligible, but impact on the spatial distribution of carbon dioxide uptake and release across regions and continents is significant. To estimate the consumption and release of carbon by humans over the landscape, we developed a carbon budget for humans in the United States. The budget was derived from food commodity intake data for the US and from algorithms representing the metabolic processing of carbon by humans. Data on consumption, respiration, and waste of carbon by humans were distributed over the US using geospatial population data with a resolution of ~450 × 450 m. The average adult in the US contains about 21 kg C and consumes about 67 kg C year⁻¹ which is balanced by the annual release of about 59 kg C as expired CO₂, 7 kg C as feces and urine, and less than 1 kg C as flatus, sweat, and aromatic compounds. In 2000, an estimated 17.2 Tg C were consumed by the US population and 15.2 Tg C were expired to the atmosphere as CO₂. Historically, carbon stock in the US human population has increased between 1790 and 2006 from 0.06 Tg to 5.37 Tg. Displacement and release of total harvested carbon per capita in the US is nearly 12% of per capita fossil fuel emissions. Humans are using, storing, and transporting carbon about the Earth’s surface. Inclusion of these carbon dynamics in regional carbon budgets can improve our understanding of carbon sources and sinks.