ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation.

ApoA-I, the main protein component of HDL, is suggested to be involved in metabolic homeostasis. We examined the effects of Milano, a naturally occurring ApoA-I variant, about which little mechanistic information is available. Remarkably, high-fat-fed mice treated with Milano displayed a rapid weight loss greater than ApoA-I WT treated mice, and a significantly reduced adipose tissue mass, without an inflammatory response. Further, lipolysis in adipose cells isolated from mice treated with either WT or Milano was increased. In primary rat adipose cells, Milano stimulated cholesterol efflux and increased glycerol release, independently of ß-adrenergic stimulation and phosphorylation of hormone sensitive lipase (Ser563) and perilipin (Ser522). Stimulation with Milano had a significantly greater effect on glycerol release compared with WT but similar effect on cholesterol efflux. Pharmacological inhibition or siRNA silencing of ABCA1 did not diminish Milano-stimulated lipolysis, although binding to the cell surface was decreased, as analyzed by fluorescence microscopy. Interestingly, methyl-ß-cyclodextrin, a well-described cholesterol acceptor, dose-dependently stimulated lipolysis. Together, these results suggest that decreased fat mass and increased lipolysis following Milano treatment in vivo is partly explained by a novel mechanism at the adipose cell level comprising stimulation of lipolysis independently of the canonical cAMP/protein kinase A signaling pathway.

Single injections of apoA-I acutely improve in vivo glucose tolerance in insulin-resistant mice.

AIMS/HYPOTHESIS: Apolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys). METHODS: Male C57Bl6 mice on a high-fat diet for 2 weeks that received a single injection of human apoA-I proteins (wild-type and Milano) were analysed for blood glucose and insulin levels during a 3 h incubation followed by glucose tolerance tests. Incorporation of injected human apoA-I protein into HDLs was analysed by native gel electrophoresis. RESULTS: ApoA-I treatment significantly improved insulin secretion and blood glucose clearance in the glucose tolerance test, with an efficiency exceeding that of lean control animals, and led to decreased basal glucose during the 3 h incubation. Notably, the two apoA-I variants triggered insulin secretion and glucose clearance to the same extent. CONCLUSIONS/INTERPRETATION: ApoA-I treatment leads to insulin- and non-insulin-dependent effects on glucose homeostasis. The experimental model of short-term (2 weeks) feeding of a high-fat diet to C57Bl6 mice provides a suitable and time-efficient system to unravel the resulting tissue-specific mechanisms of acute apoA-I treatment that lead to improved glucose homeostasis.

New insights into complex formation by SARS-CoV-2 nsp10 and nsp14.

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.

Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS).

The baculovirus expression vector system (BEVS) is one of the most popular eukaryotic systems for recombinant protein production. The focus of our protein production platform is the provision of recombinant proteins for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. Here, we present methods that reflect our standard operating procedures and setup to be able to frequently, and often repeatedly, produce many different types of proteins.

Protein production, kinetic and biophysical characterization of three human dihydroorotate dehydrogenase mutants associated with Miller syndrome.

Miller syndrome is a rare Mendelian disorder caused by mutations in the gene encoding human dihydroorotate dehydrogenase (DHODH). Human DHODH, a Class II DHODH, is an integral protein of the inner mitochondrial membrane (IMM) catalyzing the fourth step of the de novo pyrimidine biosynthesis pathway. Here we present a summary of the state of knowledge regarding Miller syndrome in the absence of any current review on the topic. We then describe the production and characterization of three distinct DHODH missense mutations (G19E, E52G, R135C) associated with Miller syndrome by means of enzyme kinetics and biophysical techniques. These human DHODH mutants were produced both in E. coli and in insect cells using the baculovirus expression vector system. We can show that the effects of these mutations differ from each other and the wild-type enzyme with respect to decreased enzymatic activity, decreased protein stability and probably disturbance of the correct import into the IMM. In addition, our results show that the N-terminus of human DHODH is not only a structural element necessary for correct mitochondrial import and location of DHODH on the outer side of the IMM, but also influences thermal stability, enzymatic activity and affects the kinetic parameters.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2023749 .

Preparation of human dihydroorotate dehydrogenase for interaction studies with lipid bilayers.

Human dihydroorotate dehydrogenase (DHODH) is an integral protein of the inner mitochondrial membrane (IMM) that catalyzes the fourth step of the de novo pyrimidine biosynthesis and is functionally connected to the respiratory chain via its lipophilic co-substrate, ubiquinone Q10. DHODH is the target for drugs approved for the treatment of rheumatoid arthritis and multiple sclerosis, and mutations in its sequence have been identified as the cause of Miller syndrome, a rare genetic disorder. The N-terminus of DHODH consists of a signal peptide for mitochondrial import (MS), a transmembrane domain (TM), followed by a microdomain which interacts with the lipids of the IMM and has been proposed to form the binding site for ubiquinone Q10. However, the mechanism by which DHODH interacts with the membrane-embedded Q10 and the lipids of the IMM remains unknown. We present the preparation and characterization of proteins necessary for investigating the structural interactions of DHODH with the lipids of the IMM, including expression and purification of full-length and N-terminally truncated (without MS and TM) DHODH. We characterized the interaction of truncated DHODH with lipid bilayers containing some key lipids of the IMM using Quartz Crystal Microbalance with Dissipation monitoring and compared it to the DHODH from E. coli, a DHODH that naturally lacks a TM. Our results suggest that although cardiolipin enhances the interaction of truncated DHODH with lipid bilayers, the presence of the TM in human DHODH is necessary for stable binding to and securing its location at the outer surface of the IMM.

[Major renal biopsy complications in 11-year-old boy--a case report]. / Ciezkie powiklania biopsji nerki u 11-letniego chlopca--opis przypadku.

Renal biopsy is an essential procedure in the diagnosis, treatment and follow-up of renal diseases. The risks associated with performing percutaneous renal biopsy have substantially decreased in the past decades. However, major complications still occur. We report and discuss a case of severe renal biopsy complications in 11-year-old with nephrotic syndrome.

[Natural history of fetal/newborn ovarian cyst]. / Historia naturalna plodowych/noworodkowych torbieli jajnika.

OBJECTIVE: Fetal and neonatal ovarian cysts are detected freguently by ultrasonography. Prenatal ovarian cysts have a follicular or luteal origin. Their natural history is a spontaneous involution. Pre- and postnatal changes in the sonographic aspects allow to choose the appropriate therapeutical indications: conservative approach, surgery, or percutaneous aspiration. MATERIALALS AND METHODS: In retrospective review from 1992-2004 authors present 26 patients with ovarian cysts. RESULTS: Fifteen patients were diagnosed by prenatal ultrasonography carried out between 33 and 39 weeks of gestation. Postnatally 14 were observed only and their cysts regressed, 10 with cysts larger than 4 cm, had surgical exploration, 2 patients with cysts between 2 to 4 cm were candidates for percutaneous aspiration. CONCLUSION: 1. The conservative treatment and clinical observation using ultrasonography is recommended when the ovari cystic is 40 mm in diameter. 2. When the ovari cystic is bigger then 40 mm in diameter it is recommended to perform puncture and fluid aspiration from the cystis under ultrasonography control. 3. The surgical intervention was planned when the ovari cystis was heterogenous and has been growing for a few months.

Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities.

"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the beta-sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13CH2D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.

[Rare coexistence of phlegmonous appendicitis and tumor of a twisted left ovary]. / Rzadkie wspólistnienie ropnego zapalenia wyrostka robaczkowego i skreconego guza lewego jajnika.

The authors present a diagnostically difficult case of a three year old girl with abdominal pain. The girl with abdominal pains, nausea, upper airways infarction and some urinary system symptoms was admitted to Children's Surgical Clinic for observation. She was given antibiotic therapy and i.v. infusions. WBC was 29.6 tys./ul and CRP 2.7 mg/dl. No other abnormalities were detected in biochemical or sonographic investigation. The girl was submitted to laparotomy because of unclear abdominal signs suggesting acute appendicitis. Phlegmonous appendicitis and twisted/rotated left ovary with multiple adhesions were found. Histopathological investigation showed teratoma of the left ovary. Postoperative course went without complication.

Secondary structure changes in ApoA-I Milano (R173C) are not accompanied by a decrease in protein stability or solubility.

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein (HDL) and a principal mediator of the reverse cholesterol transfer pathway. Variants of apoA-I have been shown to be associated with hereditary amyloidosis. We previously characterized the G26R and L178H variants that both possess decreased stability and increased fibril formation propensity. Here we investigate the Milano variant of apoAI (R173C; apoAI-M), which despite association with low plasma levels of HDL leads to low prevalence of cardiovascular disease in carriers of this mutation. The R173C substitution is located to a region (residues 170 to 178) that contains several fibrillogenic apoA-I variants, including the L178H variant, and therefore we investigated a potential fibrillogenic property of the apoAI-M protein. Despite the fact that apoAI-M shared several features with the L178H variant regarding increased helical content and low degree of ThT binding during prolonged incubation in physiological buffer, our electron microscopy analysis revealed no formation of fibrils. These results suggest that mutations inducing secondary structural changes may be beneficial in cases where fibril formation does not occur.

Comparison between the backbone dynamics of an 11-amino acid peptide sequence in alpha-helical and beta-hairpin structural contexts.

To investigate the relationship between backbone motions and the structural environment of a peptide sequence, we have used (15)N NMR relaxation data to characterize the backbone motions of the "chameleon-alpha" (Chm-alpha) and "chameleon-beta" (Chm-beta) proteins designed previously by Minor and Kim [Minor, D. L., Jr., and Kim, P. S. (1996) Nature 380, 730-734]. These two proteins contain an identical 11-amino acid sequence (dubbed the "chameleon" peptide sequence) in alpha-helix and beta-hairpin conformations, respectively, within the B1 domain of protein G. When placed in an alpha-helical context, the chameleon peptide shows very limited backbone motions, but some remote regions of the protein are induced to undergo conformational exchange motions, apparently due to modification of packing interactions with the chameleon peptide. In contrast, within a beta-hairpin context, the chameleon peptide displays substantial motions on both picosecond and microsecond-to-millisecond time scales, suggesting that it cannot be readily accommodated within the native reverse turn structure. These observations are consistent with the relatively low stability of the Chm-beta protein and can be rationalized in terms of native turn-stabilizing interactions that may be disrupted in the Chm-beta protein.