Further characterization of mitochondrial protein phosphatase.

Mitochondrial protein phosphatase from rat liver exhibits rather wide substrate specificity, since it readily dephosphorylates, besides phosvitin, casein, and cytosol phosphoproteins, also ATP, ADP, inorganic pyrophosphate, p-nitrophenylphosphate.Aliphatic phosphate esters (β-glycerophosphate, glucose-6-P, serine-phosphate) are not dephosphorylated to any detectable extent.Evidence for the participation of a single enzyme in the dephosphorylation of phosvitin and ATP is provided. However, the different affinity toward the two substrates and other evidence suggest that the enzyme has in vivo the biological role of dephosphorylating, at least preferentially, the phosphoproteins.     

Fatigue of the plantar intrinsic foot muscles increases navicular drop

Our purpose was to assess the effect of foot intrinsic muscle fatigue on pronation, as assessed with navicular drop, during static stance. Twenty-one healthy young adults participated. Navicular drop was measured before and after fatiguing exercise of the plantar foot intrinsic muscles. Surface electromyography of the abductor hallucis muscle was recorded during maximum voluntary isometric contractions (MVIC) in order to find the baseline median frequency (MedF). Subjects then performed sets of 75 repetitions of isotonic flexion contractions of the intrinsic foot muscles against a 4.55 kg weight on a custom pulley system. After each set an MVIC was performed to track shifts in MedF. After a MedF shift of at least 10%, navicular drop measurements were repeated. Subjects exhibited 10.0 ± 3.8 mm of navicular drop at baseline and 11.8 ± 3.8 mm after fatigue (p < 0.0005). The change in navicular drop was significantly correlated with change in MedF (r = .47, p = .03). The intrinsic foot muscles play a role in support of the medial longitudinal arch in static stance. Disrupting the function of these muscles through fatigue resulted in an increase in pronation as assessed by navicular drop.     

X-ray magnetic circular dichroism investigation of the superparamagnetic transition-metal ion-cluster r-Mn12Bz

We present the results of an X-ray magnetic circular dichroism investigation of a reduced version of Mn₁₂ benzoate. At variance with the parent Mn₁₂ benzoate compound, which, analogously to Mn₁₂ acetate, has a ground-state spin equal to ten, the reduced species has a ground-state with total spin S = 19/2. The half-integer spin in the ground-state makes this compound an appealing system where to test parity effects on the efficiency of the quantum tunnelling of the magnetisation. We exploited the sensitivity of X-ray magnetic circular dichroism to the oxidation state of the absorbing metal ion to obtain information about the internal structure of the reduced Mn₁₂ benzoate. In particular, we performed multiplet calculations to analyse the X-ray magnetic circular dichroism spectra at the manganese L_2,3 edge and identify the contribution of the Mn^II ion resulting by the reduction process.     

An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A

The synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.     

Preferential dephosphorylation of protein bound phosphorylthreonine and phosphorylserine residues by cytosol and mitochondrial "casein phosphatases".

The partial purification of a rat liver cytosol protein phosphatase is described, resulting in a preparation active on casein but not on phosvitin, cytosol phosphopeptides, ATP, ADP and p-nitrophenylphosphate, which, on the contrary, are still dephosphorylated by the protein phosphatase purified from rat liver mitochondria. Moreover the activity of the former enzyme on casein appears to involve only a limited amount of phosphoric sites which are also preferentially phosphorylated by soluble protein kinase. The isolation and evaluation of ³²P-serine and ³²P-threonine from protein-kinase-dependently labelled phosvitin and casein, before and after incubation with the two enzymes, led to the conclusion that mitochondrial protein phosphatase hydrolyzes more actively the phosphorylserine residues, while the cytosol “casein phosphatase” promotes a preferential breakdown of the ³²P-threonine residues.     

Protease-activated receptor 1 as a potential therapeutic target for COVID-19

Acute respiratory disease caused by a novel coronavirus (SARS-CoV-2) has spread all over the world, since its discovery in 2019, Wuhan, China. This disease is called COVID-19 and already killed over 1 million people worldwide. The clinical symptoms include fever, dry cough, dyspnea, headache, dizziness, generalized weakness, vomiting, and diarrhea. Unfortunately, so far, there is no validated vaccine, and its management consists mainly of supportive care. Venous thrombosis and pulmonary embolism are highly prevalent in patients suffering from severe COVID-19. In fact, a prothrombotic state seems to be present in most fatal cases of the disease. SARS-CoV-2 leads to the production of proinflammatory cytokines, causing immune-mediated tissue damage, disruption of the endothelial barrier, and uncontrolled thrombogenesis. Thrombin is the key regulator of coagulation and fibrin formation. In severe COVID-19, a dysfunctional of physiological anticoagulant mechanisms leads to a progressive increase of thrombin activity, which is associated with acute respiratory distress syndrome development and a poor prognosis. Protease-activated receptor type 1 (PAR1) is the main thrombin receptor and may represent an essential link between coagulation and inflammation in the pathophysiology of COVID-19. In this review, we discuss the potential role of PAR1 inhibition and regulation in COVID-19 treatment.