Evolutionary Divergence of Duplicate Copies of the Growth Hormone Gene in Suckers (Actinopterygii: Catostomidae)

Catostomid fishes (suckers) have duplicate copies of the growth hormone gene and other nuclear genes, due to a genome duplication event early in the group’s history. Yet, paralogs of GH in suckers are more than 90% conserved in nucleotide (nt) and amino acid (aa) sequence. Within paralogs across species, variation in nt and aa sequence averages 3.33% and 4.46% for GHI, and 3.22% and 2.43% for GHII, respectively. Selection tests suggest that the two GH paralogs are under strong purifying selection. Consensus trees from phylogenetic analysis of GH coding region data for 23 species of suckers, other cypriniform fishes and outgroups resolved cypriniform relationships and relationships among GHI sequences of suckers more or less consistently with analyses based on other molecular data. However, the analysis failed to resolve all sucker GHI and GHII sequences as monophyletic sister groups. This unexpected topology did not differ significantly from topologies constrained to make all GH sequences monophyletic. We attribute this result either to limitations in our GHII data set or convergent adaptive changes in GHII of tribe Catostomini

NEW HYPOTHESES ABOUT THE STRUCTURE-FUNCTION OF PCSK9, PART 1: ANALYSIS OF THE EGF-A DOCKING SITE USING WATERMAP

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to and targets the low density lipoprotein receptor (LDL-R) for lysozomal degradation. Down regulation of LDL-R leads to dyslipidemia and cardiovascular disease, whereas the beneficial effects of PCSK9 mediated regulation of LDL-R are currently unknown. We used Biacore to measure binding between PCSK9 and LDL-R, and between PCSK9 and the epidermal growth factor-like repeat A (EGF-A) of LDL-R. EGF-A exhibits fast kon and fast koff, whereas receptor binding and unbinding are slower. We also measured binding between PCSK9 and EGF-A using isothermal titration calorimetry (ITC). Heat transfer was not detected, consistent with an entropically driven process. We analyzed and compared the crystal structures of wild type vs. mutant (Asp374Tyr) PCSK9 bound to EGF-A using a new capability called WaterMap, which predicts the energies and positions of protein hydration sites. EGF-A was found to rely heavily on stable hydration sites for binding, placing polar atoms at these positions. Entropic gain, and fast kon are expected under these conditions. Insufficient availability of unstable hydration sites needed to slow EGF-A unbinding explains the relatively fast koff observed. The considerably slower koff observed for PCSK9-LDL-R unbinding is consistent with the existence of additional unstable hydration sites at a second docking site outside of EGF-A. WaterMap further suggests that the slower koff observed for Asp374Tyr PCSK9 is due to a net gain of unstable hydration sites resulting from this mutation. We hypothesize that endosomal pH speeds up kon for both LDL-R and EGF-A due to a gain of additional stable hydration sites at or near the EGF-A interface, and slows koff for LDL-R due to gain of additional unstable hydration sites

Pharmacological Inhibition of Stearoyl CoA Desaturase 1 in the Skin Induces Atrophy of the Sebaceous Glands

Stearoyl-CoA desaturase-1 (SCD-1) catalyzes the formation of delta9-monounsaturated fatty acids from saturated precursors. Deficiency of SCD-1 in mice causes atrophy of sebaceous glands. We asked whether local pharmacological inhibition of this enzyme in the skin would lead to a similar effect. To this end, we characterized the low-molecular-weight compound XEN103 as a potent and selective inhibitor of SCD-1 activity. The compound blocks microsomal and cellular SCD-1 activity across species with IC50 values in the range of 10-15 nM. Upon topical application to the skin of mice as a 1% solution, XEN103 induces pronounced sebaceous gland atrophy with a rapid onset after a few days of dosing, both sebaceous gland numbers and size being reduced by 50 to 75%, and without any signs of skin irritation. We show that the effect is due to the local action of the compound on SCD-1 in the skin and occurs in the presence of only minimal plasma exposure. Based on these observations, SCD-1 inhibitors such as XEN103 may provide an opportunity to develop a novel topical therapy for acne, as a disease characterized by overproduction of sebum from hypertrophic sebaceous glands

New hypotheses about the structure–function of proprotein convertase subtilisin/kexin type 9: Analysis of the epidermal growth factor-like repeat A docking site using WaterMap

LDL cholesterol (LDL-C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low-density lipoprotein cholesterol receptor (LDL-R). LDL-R is targeted for lysosomal degradation by association with proprotein convertase subtilisin-kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor-sparing phenomena, including loss-of-function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate-determining steps for protein–ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild-type versus gain-offunction mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins (‘‘hydration sites’’). We compared these results with binding kinetics data for PCSK9, full-length LDL-R ectodomain, and isolated EGF-A repeat. We propose that the fast kon and entropically driven thermodynamics observed for PCSK9EGF-A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H-bonds from water to polar EGF-A groups). We further propose that the relatively fast koff observed for EGFA unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower koff observed for EGF-A and LDL-R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex

LLF580, an FGF21 Analog, Reduces Triglycerides and Hepatic Fat in obese persons with modestly elevated triglycerides: A 12 Week Randomized Double Blind Placebo Controlled Study

Purpose: To evaluate the safety and potential efficacy of LLF580, a genetically engineered variant of human fibroblast growth factor-21, for triglyceride lowering, weight loss, and hepatic fat reduction. Methods: A multicenter, double-blind, parallel design trial in obese, mildly hypertriglyceridemic and otherwise apparently healthy adults randomized (1:1) to LLF580 300 mg or placebo subcutaneously every four weeks for three doses. Results: Of 64 randomized study participants, 61 (mean±SD: age 45±11 years, 49% male, 80/15/5% Caucasian/African American/Other, BMI 36.1±3.8 kg/m2) received LLF580 (n=30) or placebo (n=31) at 7 research sites in the USA. LLF580 lowered serum triglycerides by 54% (least square mean placebo adjusted change from baseline), total cholesterol 7%, LDL-cholesterol 12%, and increased HDL-cholesterol 36% compared to placebo (all P<0.001) over 12 weeks. Substantial reduction of liver fat of 52% over placebo (P<0.001) was also demonstrated, in the setting of improved liver function tests including alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, the composite enhanced liver fibrosis score, and N-terminal type III collagen propeptide (all P<0.05). Insulin and C-peptide levels and insulin resistance by HOMA-IR were all lower, and adiponectin higher with LLF580 treatment compared to placebo, while fasting glucose and HbA1c were unchanged. Reductions in biomarkers of bone formation without differences in markers of bone resorption were observed. LLF580 was generally safe and well tolerated, except for higher incidence of generally mild to moderate gastrointestinal adverse effects. Conclusions: In obese, mildly hypertriglyceridemic adults, LLF580 was generally safe and demonstrated beneficial effects on serum lipids, liver fat and biomarkers of liver injury, suggesting it may be effective for treatment of select metabolic disorders including hypertriglyceridemia and non-alcoholic fatty liver disease. Assessments of longer-term safety and efficacy are warranted

Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity.

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation

LLF580, an FGF21 Analog, Reduces Triglycerides and Hepatic Fat in obese persons with modestly elevated triglycerides: A 12 Week Randomized Double Blind Placebo Controlled Study

Purpose: To evaluate the safety and potential efficacy of LLF580, a genetically engineered variant of human fibroblast growth factor-21, for triglyceride lowering, weight loss, and hepatic fat reduction. Methods: A multicenter, double-blind, parallel design trial in obese, mildly hypertriglyceridemic and otherwise apparently healthy adults randomized (1:1) to LLF580 300 mg or placebo subcutaneously every four weeks for three doses. Results: Of 64 randomized study participants, 61 (mean±SD: age 45±11 years, 49% male, 80/15/5% Caucasian/African American/Other, BMI 36.1±3.8 kg/m2) received LLF580 (n=30) or placebo (n=31) at 7 research sites in the USA. LLF580 lowered serum triglycerides by 54% (least square mean placebo adjusted change from baseline), total cholesterol 7%, LDL-cholesterol 12%, and increased HDL-cholesterol 36% compared to placebo (all P<0.001) over 12 weeks. Substantial reduction of liver fat of 52% over placebo (P<0.001) was also demonstrated, in the setting of improved liver function tests including alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, the composite enhanced liver fibrosis score, and N-terminal type III collagen propeptide (all P<0.05). Insulin and C-peptide levels and insulin resistance by HOMA-IR were all lower, and adiponectin higher with LLF580 treatment compared to placebo, while fasting glucose and HbA1c were unchanged. Reductions in biomarkers of bone formation without differences in markers of bone resorption were observed. LLF580 was generally safe and well tolerated, except for higher incidence of generally mild to moderate gastrointestinal adverse effects. Conclusions: In obese, mildly hypertriglyceridemic adults, LLF580 was generally safe and demonstrated beneficial effects on serum lipids, liver fat and biomarkers of liver injury, suggesting it may be effective for treatment of select metabolic disorders including hypertriglyceridemia and non-alcoholic fatty liver disease. Assessments of longer-term safety and efficacy are warranted