The conformation of human phospholipid scramblase 1, as studied by infrared spectroscopy. Effects of calcium and detergent.

Human phospholipid scramblase 1 (SCR) is a membrane protein that catalyzes the transmembrane (flip-flop) motion of phospholipids. It can also exist in a non membrane-bound form in the nucleus, where it modulates several aspects of gene expression. Catalysis of phospholipid flip-flop requires the presence of millimolar Ca2+, and occurs in the absence of ATP. Membrane-bound SCR contains a C-terminal α-helical domain embedded in the membrane bilayer. The latter domain can be removed giving rise to a stable truncated mutant SCRΔ that is devoid of scramblase activity. In order to improve our understanding of SCR structure infrared spectra have been recorded of both the native and truncated forms, and the effects of adding Ca2+, or removing detergent, or thermally denaturing the protein have been observed. Under all conditions the main structural component of SCR/SCRΔ is a ß-sheet. Removing the C-terminal 28 aa residues, which anchor SCR to the membrane, leads to a change in tertiary structure and an increased structural flexibility. The main effect of Ca2+ is an increase in the α/ß ratio of secondary structure components, with a concomitant increase in the proportion of non-periodic structures. At least in SCRΔ, detergent (Zwittergent 3-12) decreases the structural flexibility, an effect somewhat opposite to that of increasing temperature. Thermal denaturation is affected by Ca2+, detergent, and by the presence or absence of the C-terminal domain, each of them influencing in different ways the denaturation pattern.

The C-terminal transmembrane domain of human phospholipid scramblase 1 is essential for the protein flip-flop activity and Ca²âº-binding.

Human phospholipid scramblase 1 (SCR) is a 318 amino acid protein that was originally described as catalyzing phospholipid transbilayer (flip-flop) motion in plasma membranes in a Ca²âº-dependent, ATP-independent way. Further studies have suggested an intranuclear role for this protein in addition. A putative transmembrane domain located at the C terminus (aa 291-309) has been related to the flip-flop catalysis. In order to clarify the role of the C-terminal region of SCR, a mutant was produced (SCRΔ) in which the last 28 amino acid residues were lacking, including the α-helix. SCRΔ had lost the scramblase activity and its affinity for Ca²âº was decreased by one order of magnitude. Fluorescence and IR spectroscopic studies revealed that the C-terminal region of SCR was essential for the proper folding of the protein. Moreover, it was found that Ca²âº exerted an overall destabilizing effect on SCR, which might facilitate its binding to membranes.

Binding of ß-amyloid (1-42) peptide to negatively charged phospholipid membranes in the liquid-ordered state: modeling and experimental studies.

To explore the initial stages of amyloid ß peptide (Aß42) deposition on membranes, we have studied the interaction of Aß42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L(o)) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L(o) state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5-5 mol%) than higher (10-20 mol%) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aß42 binding than L(o) ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of ß-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aß42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol% anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aß42 and membrane.

Analysis on the Characterization of Multiphoton Microscopy Images for Malignant Neoplastic Colon Lesion Detection under Deep Learning Methods.

BACKGROUND: Colorectal cancer has a high incidence rate worldwide, with over 1.8 million new cases and 880,792 deaths in 2018. Fortunately, its early detection significantly increases the survival rate, reaching a cure rate of 90% when diagnosed at a localized stage. Colonoscopy is the gold standard technique for detection and removal of colorectal lesions with potential to evolve into cancer. When polyps are found in a patient, the current procedure is their complete removal. However, in this process, gastroenterologists cannot assure complete resection and clean margins which are given by the histopathology analysis of the removed tissue, which is performed at laboratory. AIMS: In this paper, we demonstrate the capabilities of multiphoton microscopy (MPM) technology to provide imaging biomarkers that can be extracted by deep learning techniques to identify malignant neoplastic colon lesions and distinguish them from healthy, hyperplastic, or benign neoplastic tissue, without the need for histopathological staining. MATERIALS AND METHODS: To this end, we present a novel MPM public dataset containing 14,712 images obtained from 42 patients and grouped into 2 classes. A convolutional neural network is trained on this dataset and a spatially coherent predictions scheme is applied for performance improvement. RESULTS: We obtained a sensitivity of 0.8228 ± 0.1575 and a specificity of 0.9114 ± 0.0814 on detecting malignant neoplastic lesions. We also validated this approach to estimate the self-confidence of the network on its own predictions, obtaining a mean sensitivity of 0.8697 and a mean specificity of 0.9524 with the 18.67% of the images classified as uncertain. CONCLUSIONS: This work lays the foundations for performing in vivo optical colon biopsies by combining this novel imaging technology together with deep learning algorithms, hence avoiding unnecessary polyp resection and allowing in situ diagnosis assessment.

Mechanistic Contributions of Biological Cofactors in Islet Amyloid Polypeptide Amyloidogenesis.

Type II diabetes mellitus is associated with the deposition of fibrillar aggregates in pancreatic islets. The major protein component of islet amyloids is the glucomodulatory hormone islet amyloid polypeptide (IAPP). Islet amyloid fibrils are virtually always associated with several biomolecules, including apolipoprotein E, metals, glycosaminoglycans, and various lipids. IAPP amyloidogenesis has been originally perceived as a self-assembly homogeneous process in which the inherent aggregation propensity of the peptide and its local concentration constitute the major driving forces to fibrillization. However, over the last two decades, numerous studies have shown a prominent role of amyloid cofactors in IAPP fibrillogenesis associated with the etiology of type II diabetes. It is increasingly evident that the biochemical microenvironment in which IAPP amyloid formation occurs and the interactions of the polypeptide with various biomolecules not only modulate the rate and extent of aggregation, but could also remodel the amyloidogenesis process as well as the structure, toxicity, and stability of the resulting fibrils.

A conventional an 2DCOS infrared approach to the kinetics of protein misfolding.

Cell viability depends on the correct folding of the proteins involved in metabolism. Proteins are synthesized on the endoplasmic reticulum and must follow a pathway to a correct, metastable, tridimensional structure. Changes in structure or in environmental conditions can drive an instability of the folding conditions and produce non-active aggregates that in principle are proteolysed by the cellular mechanisms. However, these aggregates can be even more stable than the native proteins, escaping the cellular control. They can be classified as amorphous, if there is not a well-organized structural pattern, or ordered if a repetitive pattern is produced. These ordered structures, known as fibrils, are involved in many diseases. Infrared spectroscopy is a method of choice to study its formation because it is not affected by turbidity or the formation of high molecular weight aggregates. Moreover, in both cases, two bands characteristic of intermolecular ß-sheets allow the monitoring of the aggregate formation. In both cases, the appearance of these bands involves a non-reversible path in protein folding. It has been suggested that a difference in the ordered structures involves an increasing in band intensity. This change can be the origin in variations on the 2DCOS maps. The synchronous map gives an overall idea of the process involved. The asynchronous is more informative because reflects the kinetic changes produced. The outcome of both processes, amorphous or ordered is that 2DCOS can provide a further insight to the knowledge of the kinetic processes giving rise to aggregated structures. This outcome could consist on the order in which the different secondary structures are prone to form the aggregates.