Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux.

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.

The Effect of Blood Flow Restriction Therapy on Shoulder Function Following Shoulder Stabilization Surgery: A Case Series.

Background: Traumatic shoulder instability is a common injury in athletes and military personnel. Surgical stabilization reduces recurrence, but athletes often return to sport before recovering upper extremity rotational strength and sport-specific abilities. Blood flow restriction (BFR) may stimulate muscle growth without the need for heavy resistance training post-surgically. Hypothesis/Purpose: To observe changes in shoulder strength, self-reported function, upper extremity performance, and range of motion (ROM) in military cadets recovering from shoulder stabilization surgery who completed a standard rehabilitation program with six weeks of BFR training. Study Design: Prospective case series. Methods: Military cadets who underwent shoulder stabilization surgery completed six weeks of upper extremity BFR training, beginning post-op week six. Primary outcomes were shoulder isometric strength and patient-reported function assessed at 6-weeks, 12-weeks, and 6-months postoperatively. Secondary outcomes included shoulder ROM assessed at each timepoint and the Closed Kinetic Chain Upper Extremity Stability Test (CKCUEST), the Upper Extremity Y-Balance Test (UQYBT), and the Unilateral Seated Shotput Test (USPT) assessed at the six-month follow-up. Results: Twenty cadets performed an average 10.9 BFR training sessions over six weeks. Statistically significant and clinically meaningful increases in surgical extremity external rotation strength (p < 0.001; mean difference, .049; 95% CI: .021, .077), abduction strength (p < 0.001; mean difference, .079; 95% CI: .050, .108), and internal rotation strength (p < 0.001; mean difference, .060; CI: .028, .093) occurred from six to 12 weeks postoperatively. Statistically significant and clinically meaningful improvements were reported on the Single Assessment Numeric Evaluation (p < 0.001; mean difference, 17.7; CI: 9.4, 25.9) and Shoulder Pain and Disability Index (p < 0.001; mean difference, -31.1; CI: -44.2, -18.0) from six to 12 weeks postoperatively. Additionally, over 70 percent of participants met reference values on two to three performance tests at 6-months. Conclusion: While the degree of improvement attributable to the addition of BFR is unknown, the clinically meaningful improvements in shoulder strength, self-reported function, and upper extremity performance warrant further exploration of BFR during upper extremity rehabilitation. Level of Evidence: 4, Case Series.

Modified sites and functional consequences of 4-oxo-2-nonenal adducts in HDL that are elevated in familial hypercholesterolemia.

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.

A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL.

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins.

Cross-linking coupled with mass spectrometry (XL-MS) has emerged as a powerful strategy for the identification of protein-protein interactions, characterization of interaction regions, and obtainment of structural information on proteins and protein complexes. In XL-MS, proteins or complexes are covalently stabilized with cross-linkers and digested, followed by identification of the cross-linked peptides by tandem mass spectrometry (MS/MS). This provides spatial constraints that enable modeling of protein (complex) structures and regions of interaction. However, most XL-MS approaches are not capable of differentiating intramolecular from intermolecular links in multimeric complexes, and therefore they cannot be used to study homodimer interfaces. We have recently developed an approach that overcomes this limitation by stable isotope-labeling of one of the two monomers, thereby creating a homodimer with one 'light' and one 'heavy' monomer. Here, we describe a step-by-step protocol for stable isotope-labeling, followed by controlled denaturation and refolding in the presence of the wild-type protein. The resulting light-heavy dimers are cross-linked, digested, and analyzed by mass spectrometry. We show how to quantitatively analyze the corresponding data with SIM-XL, an XL-MS software with a module tailored toward the MS/MS data from homodimers. In addition, we provide a video tutorial of the data analysis with this protocol. This protocol can be performed in ∼14 d, and requires basic biochemical and mass spectrometry skills.

Plzf is required in adult male germ cells for stem cell self-renewal.

Adult germline stem cells are capable of self-renewal, tissue regeneration and production of large numbers of differentiated progeny. We show here that the classical mouse mutant luxoid affects adult germline stem cell self-renewal. Young homozygous luxoid mutant mice produce limited numbers of normal spermatozoa and then progressively lose their germ line after birth. Transplantation studies showed that germ cells from mutant mice did not colonize recipient testes, suggesting that the defect is intrinsic to the stem cells. We determined that the luxoid mutant contains a nonsense mutation in the gene encoding Plzf, a transcriptional repressor that regulates the epigenetic state of undifferentiated cells, and showed that Plzf is coexpressed with Oct4 in undifferentiated spermatogonia. This is the first gene shown to be required in germ cells for stem cell self-renewal in mammals.

Reproductive aging and variability in the ovarian antral follicle count: application in the clinical setting.

OBJECTIVE: To determine the extent of intercycle and interobserver variability in antral follicle (AF) count and their impact on stimulation quality in IVF. DESIGN: Prospective evaluation of the impact on AF count of GnRH agonist down-regulation and interobserver variability. Retrospective evaluation of intercycle variability in AF count. SETTING: University ART clinic. PATIENT(S): Twenty subjects were used to evaluate the effect of GnRH agonist down-regulation upon AF count; six of whom were used to evaluate interobserver variability. Fifty patients experiencing two or three cycles of IVF within a 1-year interval. INTERVENTION(S): Transvaginal ultrasound exams before and after down-regulation with a GnRH agonist. Videotaped day-3 transvaginal ultrasound exams. MAIN OUTCOME MEASURE(S): [1] Intercycle and interobserver variability in antral follicle count. [2] Oocytes retrieved, peak estradiol, gonadotropin dose, duration of stimulation and cancellation rates. RESULT(S): There is moderate intercycle and interobserver variability in AF counts. GnRH agonist down-regulation does not significantly change AF count. In infertility patients undergoing IVF, paired analysis between the low- and high-AF count cycles did not show a difference in quality of stimulation or cycle cancellation rates. CONCLUSIONS: Within an individual patient, higher AF count in a given cycle was not predictive of better stimulation compared with the case of a lower count cycle.