Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca one order of magnitude more efficiently than MICU1 and, when heterodimerized with MICU2, activates MCU current at lower Ca concentrations than MICU1-MICU2 heterodimers. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca uptake and ATP production. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca uptake machinery that allows skeletal muscle mitochondria to be highly responsive to sarcoplasmic [Ca] responses.

Background Considerable attention has been paid to perfluoroalkyl compounds (PFCs) because of their worldwide presence in humans, wildlife, and environment. A wide variety of toxicological effects is well supported in animals, including testicular toxicity and male infertility. For these reasons, the understanding of epidemiological associations and of the molecular mechanisms involved in the endocrine-disrupting properties of PFCs on human reproductive health is a major concern. Objective To investigate the relationship between PFC exposure and male reproductive health. Design This study was performed within a screening protocol to evaluate male reproductive health in high schools. Patients This is a cross-sectional study on 212 exposed males from the Veneto region, one of the four areas worldwide heavily polluted with PFCs, and 171 nonexposed controls. Main Outcome Measures Anthropometrics, seminal parameters, and sex hormones were measured in young males from exposed areas compared with age-matched controls. We also performed biochemical studies in established experimental models. Results We found that increased levels of PFCs in plasma and seminal fluid positively correlate with circulating testosterone (T) and with a reduction of semen quality, testicular volume, penile length, and anogenital distance. Experimental evidence points toward an antagonistic action of perfluorooctanoic acid on the binding of T to androgen receptor (AR) in a gene reporter assay, a competition assay on an AR-coated surface plasmon resonance chip, and an AR nuclear translocation assay. Discussion This study documents that PFCs have a substantial impact on human health as they interfere with hormonal pathways, potentially leading to male infertility.

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β2-Glycoprotein I (β2GpI) is the major autoantigen in the antiphospholipid syndrome, a thrombotic autoimmune disease. Nonetheless, the physiological role of β2GpI is still unclear. In a recent work, we have shown that β2GpI selectively inhibits the procoagulant functions of human α-thrombin (αT; i.e. prolongs fibrin clotting time, t, and inhibits αT-induced platelet aggregation) without affecting the unique anticoagulant activity of the protease, i.e. the proteolytic generation of the anticoagulant protein C (PC) from the PC zymogen, which interacts with αT exclusively at the protease catalytic site. Here, we used several different biochemical/biophysical techniques and molecular probes for mapping the binding sites in the αT-β2GpI complex. Our results indicate that αT exploits the highly electropositive exosite-II, which is also responsible for anchoring αT on the platelet GpIbα (platelet receptor glycoprotein Ibα) receptor, for binding to a continuous negative region on β2GpI structure, spanning domain IV and (part of) domain V, whereas the protease active site and exosite-I (i.e. the fibrinogen-binding site) remain accessible for substrate/ligand binding. Furthermore, we provided evidence that the apparent increase in t, previously observed with β2GpI, is more likely caused by alteration in the ensuing fibrin structure rather than by the inhibition of fibrinogen hydrolysis. Finally, we produced a theoretical docking model of αT-β2GpI interaction, which was in agreement with the experimental results. Altogether, these findings help to understand how β2GpI affects αT interactions and suggest that β2GpI may function as a scavenger of αT for binding to the GpIbα receptor, thus impairing platelet aggregation while enabling normal cleavage of fibrinogen and PC.

Trypsin-like proteases are synthesized as zymogens and activated through a mechanism that folds the active site for efficient binding and catalysis. Ligand binding to the active site is therefore a valuable source of information on the changes that accompany zymogen activation. Using the physiologically relevant transition of the clotting zymogen prothrombin to the mature protease thrombin, we show that the mechanism of ligand recognition follows selection within a pre-existing ensemble of conformations with the active site accessible (E) or inaccessible (E*) to binding. Prothrombin exists mainly in the E* conformational ensemble and conversion to thrombin produces two dominant changes: a progressive shift toward the E conformational ensemble triggered by removal of the auxiliary domains upon cleavage at R271 and a drastic drop of the rate of ligand dissociation from the active site triggered by cleavage at R320. Together, these effects produce a significant (700-fold) increase in binding affinity. Limited proteolysis reveals how the E*-E equilibrium shifts during prothrombin activation and influences exposure of the sites of cleavage at R271 and R320. These new findings on the molecular underpinnings of prothrombin activation are relevant to other zymogens with modular assembly involved in blood coagulation, complement and fibrinolysis.

Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active ␣-thrombin (␣T). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320 -Ile-321 to yield mature ␣T. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nM to 2 M), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active Pre2, a nicked Pre2 derivative with a single cleaved Ala-470 -Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470 -Asn-471 is instrumental for the onset of catalysis in Pre2, which was, however, reduced about 100 -200-fold compared with ␣T. Of note, Pre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.Blood coagulation is a finely regulated physiological process that culminates with the factor Xa-mediated conversion of the prothrombin (ProT) 3 zymogen to the active ␣-thrombin (␣T) enzyme, which in turn is responsible for the generation of insoluble fibrin and activation of platelets via the GpIb␣-PAR1 pathway (1, 2). ProT (ϳ72 kDa) is a vitamin K-dependent glycoprotein produced in the liver and circulating at a relatively high plasma concentration (0.1 mg/ml) (3). The domain architecture of ProT includes a Gla domain (residues 1-46), a kringle-1 (residues 65-143) and a kringle-2 (residues 170 -248) domain, and a chymotrypsin-like protease domain (residues 285-579) connected by three intervening linker regions (Lnk-1, -2, and -3) (4). Isolated factor Xa (FXa) has low intrinsic ProTconverting activity, but when it is assembled in the presence of Ca 2ϩ with cofactor Va in the prothrombinase complex on the platelet surface, its ability to activate ProT is increased by about 5 orders of magnitude (5). FXa cleaves ProT in a concerted manner at two sites, i.e. Arg-271 and Arg-320, but the order of peptide bond cleavage is highly context-dependent. On the platelet surface, FXa first cleaves ProT at Arg-271 generating the inactive precursor prethrombin-2 (Pre2), which is attacked by FXa at Arg-320 to generate the active ␣T species, formed by the polypeptide chains Thr-272-Arg-320 and Ile-321-Glu-579 (6). ...

Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ for their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215–217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold.