Systematic Interrogation of Protein Refolding Under Cellular-Like Conditions

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain's intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting proteins safe passage to their native states; however, the complexity of this medium has generally precluded biophysical techniques from interrogating protein folding under cellular-like conditions for single proteins, let alone entire proteomes. Here, we develop a limited-proteolysis mass spectrometry approach paired within an isotope-labeling strategy to globally monitor the structures of refolding E. coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data, and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than indirect measures like binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones both unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. The data support a model in which chaperone-nonrefolders have evolved to fold efficiently once and only once, co-translationally, and remain kinetically trapped in their native conformations.

Pathways to Parkinson’s disease: a spotlight on 14-3-3 proteins

Abstract14-3-3s represent a family of highly conserved 30 kDa acidic proteins. 14-3-3s recognize and bind specific phospho-sequences on client partners and operate as molecular hubs to regulate their activity, localization, folding, degradation, and protein–protein interactions. 14-3-3s are also associated with the pathogenesis of several diseases, among which Parkinson’s disease (PD). 14-3-3s are found within Lewy bodies (LBs) in PD patients, and their neuroprotective effects have been demonstrated in several animal models of PD. Notably, 14-3-3s interact with some of the major proteins known to be involved in the pathogenesis of PD. Here we first provide a detailed overview of the molecular composition and structural features of 14-3-3s, laying significant emphasis on their peculiar target-binding mechanisms. We then briefly describe the implication of 14-3-3s in the central nervous system and focus on their interaction with LRRK2, α-Synuclein, and Parkin, three of the major players in PD onset and progression. We finally discuss how different types of small molecules may interfere with 14-3-3s interactome, thus representing a valid strategy in the future of drug discovery.

Histochemical Evaluation of the Processes of Protein Oxidative Modification in the Extravillous Cytotrophoblast of the Utero-Placental Bed during Iron-Deficiency Anemia in Pregnancy

Utero-placental bed is the cumulation of gestationally altered endometrium at the place of ovum attachment to the uterine wall. The key mechanism of this process is the cytotrophoblastic invasion. During iron deficiency anemia, an increase in the specific volume of the extravascular invasive trophoblast is taking place. Concern for the protein oxidative modification in iron deficiency anemia is due to the fact that in conditions of hypoxia, free radical processes in the blood and tissues are enhanced, and iron deficiency is additionally able to modify this problem. The purpose of the study was to establish the histochemical features of the processes of protein oxidative modification in the fractions of extravillous cytotrophoblast of the utero-placental bed depending on the degree of iron deficiency anemia in pregnant women. Material and methods. Quantitative characteristics of protein oxidative modification in the extravillous trophoblast of the utero-placental bed of pregnant women with iron deficiency anemia by means of the histochemical method using reactions with Bromophenol Blue on “acidic” and “basic” proteins according to Mikel Calvo method and computer microdensitometry. We studied 74 biopsies of the utero-placental bed of pregnant women with iron deficiency anemia of I, II and III degrees. The term of gestation was 37-40 weeks. Results and discussion. During physiological pregnancy, the ratio between "acidic" and "basic" proteins in trophoblast cells, even normally, is characterized by a predominance of "acidic" proteins, and evenly in both intravascular and extravascular fractions of cytotrophoblast. Intensification of processes of protein oxidative modification in the cytotrophoblast of the utero-placental bed during iron deficiency anemia of I-II degrees can be assessed as moderate, with an increase in the cells of the endothelium-replacing fraction of cytotrophoblast compared with the interstitial. In conditions of anemia of III degree, a significant predominance of "acidic" proteins in the intravascular cytotrophoblast was noted. Conclusion. During the physiological pregnancy, the intensity of protein oxidative modification was equal in all fractions of the extravillous cytotrophoblast in the utero-placental bed. In the case of gestation with iron deficiency anemia, significant intensification of the protein oxidative modification in the extravillous cytotrophoblast correlated with the severity of anemia. Background iron deficiency anemia significantly affected the processes of protein oxidative modification in the endothelium-replacing cytotrophoblast

Labeling Preferences of Diazirines with Protein Biomolecules

<p>Diazirines are widely used in photoaffinity labeling (PAL) to trap non-covalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Here, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that aryl-fluorodiazirines react primarily through a carbene intermediate, while alkyl diazirines generate a reactive alkyl diazo intermediate on route to the carbene. The generation of a reactive diazo intermediate leads to preferential labeling of acidic amino acids in a pH-dependent manner. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why these probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive-charge tend to produce higher labeling yields. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome, and will facilitate probe design and interpretation of biomolecular labeling experiments with diazirines.<b></b></p>

Labeling Preferences of Diazirines with Protein Biomolecules

<p>Diazirines are widely used in photoaffinity labeling (PAL) to trap non-covalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Here, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that aryl-fluorodiazirines react primarily through a carbene intermediate, while alkyl diazirines generate a reactive alkyl diazo intermediate on route to the carbene. The generation of a reactive diazo intermediate leads to preferential labeling of acidic amino acids in a pH-dependent manner. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why these probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive-charge tend to produce higher labeling yields. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome, and will facilitate probe design and interpretation of biomolecular labeling experiments with diazirines.<b></b></p>

Autopsy Findings in Case of Fatal Scorpion Sting: A Systematic Review of the Literature

Scorpion sting is a public health issue in several countries, particularly in America, the Middle East, India and Africa. The estimated annual global incidence of scorpion envenomings is about 1.5 million, resulting in 2600 deaths. Scorpions are Arthropoda characterized by a tail ending in a terminal bulbous (telson) containing paired venom glands and the stinger. There are 19 known families of scorpions and more than 2200 species, of which about 50 from the families of Buthidae, Hemiscorpiidae and Scorpionidae are harmful to humans. Scorpion venom is a complex structure composed of neurotoxic proteins, salts, acidic proteins and organic compounds, thereby having neurologic, cardiovascular, hematologic and renal side effects, in addition to local effects such as redness, pain, burning and swelling. When the sting is fatal, the mechanism of death is often related to cardiotoxicity with terminal pulmonary edema. However, the cholinergic excess or the neuromuscular excitation can provoke respiratory failure. Sometimes, death is due to an anaphylactic reaction to the envenoming. The purpose of this literature review is to evaluate the autopsy findings in scorpion sting-related deaths in order to better understand the pathophysiological mechanisms underlying them, thus helping pathologists in defining the correct diagnosis.

Comparative Proteomics of Octocoral and Scleractinian Skeletomes and the Evolution of Coral Calcification

Corals are the ecosystem engineers of coral reefs, one of the most biodiverse marine ecosystems. The ability of corals to form reefs depends on the precipitation of calcium carbonate (CaCO3) under biological control. However, several mechanisms underlying coral biomineralization remain elusive, for example, whether corals employ different molecular machineries to deposit different CaCO3 polymorphs (i.e., aragonite or calcite). Here, we used tandem mass spectrometry (MS/MS) to compare the proteins occluded in the skeleton of three octocoral and one scleractinian species: Tubipora musica and Sinularia cf. cruciata (calcite sclerites), the blue coral Heliopora coerulea (aragonitic skeleton), and the scleractinian aragonitic Montipora digitata. Reciprocal Blast analysis revealed extremely low overlap between aragonitic and calcitic species, while a core set of proteins is shared between octocorals producing calcite sclerites. However, the carbonic anhydrase CruCA4 is present in the skeletons of both polymorphs. Phylogenetic analysis highlighted several possible instances of protein co-option in octocorals. These include acidic proteins and scleritin, which appear to have been secondarily recruited for calcification and likely derive from proteins playing different functions. Similarities between octocorals and scleractinians included presence of a galaxin-related protein, carbonic anhydrases, and one hephaestin-like protein. Although the first two appear to have been independently recruited, the third appear to share a common origin. This work represents the first attempt to identify and compare proteins associated with coral skeleton polymorph diversity, providing several new research targets and enabling both future functional and evolutionary studies aimed at elucidating the origin and evolution of coral biomineralization.

ГИСТОЛОГИЧЕСКИЕ И ЦИТОЛОГИЧЕСКИЕ ИССЛЕДОВАНИЯ ИММУННОЙ СИСТЕМЫ СТЕНКИ ТОЛСТОЙ КИШКИ У ПЛОДОВ И НОВОРОЖДЕННЫХ ТЕЛЯТ

Статья посвящена исследованиям тканей толстого кишечника в аспекте формирования его иммунной системы. У плодов 3-4,5 месяцев в слизистой оболочке слепой и ободочной кишок появляются первые рассеянные скопления лимфоцитов. У новорожденных телят в составе лимфоузелков преобладают средние и малые лимфоциты, большие лимфоциты и стромальные клетки (45,2 16,1 и 15,6). На протяжении всего плодного и новорожденного этапов развития между эпителиоцитами выявляются клетки мигранты соединительной ткани, их количество постепенно увеличивается, причем количество специфических мигрантов больше в ворсинках, чем в криптах. Гистохимическим исследованием установлено накопление в лимфоцитах мигрантах кислых белков, -SH-групп белков и рибонуклеопротеидов, выявлена закономерность развития межклеточного вещества в соединительной ткани кишечной стенки и рассмотрены этапы развития самой соединительной ткани.The article is devoted to the research of the large intestine tissues in the aspect of its immune system formation. The first open cluster of lymphocytes appear in the intestinal mucosa of the blind gut and middle gut of fetuses of 3-4, 5 months age. There is a predominance of middle and small lymphocytes, large lymphocytes and stromal cells in the lymph nodes of newborn calves (45.2, 16.1, and 15.6). During the entire fetal and newborn stages of development, migrant cells of connective tissue are detected between epithelial cells. Their number gradually increases, and the number of specific migrants is greater in the villi than in the crypts. Histochemical study determined the accumulation of acidic proteins in migrants lymphocytes, SH-groups of proteins and ribonucleoproteins identified the pattern of development of the intercellular substance in the connective tissue of the intestinal wall and considered the stages of development of connective tissue.

Comparative proteomics of octocoral and scleractinian skeletomes and the evolution of coral calcification

Corals are ecosystem engineers of the coral reefs, one of the most biodiverse but severely threatened marine ecosystems. The ability of corals to form the three dimensional structure of reefs depends on the precipitation of calcium carbonate under biologically control. However, the exact mechanisms underlying this biologically controlled biomineralization remain to be fully unelucidated, for example whether corals employ a different molecular machinery for the deposition of different calcium carbonate (CaCO3) polymorphs (i.e., aragonite or calcite). Here we used tandem mass spectrometry (MS/MS) to compare skeletogenic proteins, i.e., the proteins occluded in the skeleton of three octocoral and one scleractinian species: Tubipora musica and Sinularia cf. cruciata, both forming calcite sclerites, the blue coral Heliopora coerulea with an aragonitic rigid skeleton, and the scleractinian aragonitic Montipora digitata. We observed extremely low overlap between aragonitic and calcitic species, while a core set of proteins is shared between octocorals producing calcite sclerites. However, the same carbonic anhydrase (CruCA4) is employed for the formation of skeletons of both polymorphs. Similarities could also be observed between octocorals and scleractinians, including the presence of a galaxin-like protein. Additionally, as in scleractinians, some octocoral skeletogenic proteins, such as acidic proteins and scleritin, appear to have been secondarily co-opted for calcification and likely derive from proteins playing different extracellular functions. In H. coerulea, co-option was characterized by aspartic acid-enrichment of proteins. This work represents the first attempt to identify the molecular basis underlying coral skeleton polymorph diversity, providing several new research targets and enabling both future functional and evolutionary studies aimed at elucidating the origin and evolution of biomineralization in corals.

Experimental study of tropism in cultivated canine coronavirus in the small intestine of puppies

The varying extents of natural disease induced by coronavirus in dogs are not completely clear because the pathogenesis of coronavirus enteritis is not studied sufficiently. In this study, based on the results of clinical, virological, morphological and histochemical studies, we determined the pathogenic role of coronavirus in infected dogs using experimental infection, per os, of isolated canine coronavirus (Nick) with titer of infectious activity equaling 4.8 ± 0.04 lg TCID50/cm, cultivated on heterologous cell cultures. This allowed us to determine, supplement, and generalize the data on pathogenesis of the disease and determine the histological changes in the small intestine, where the initial replication of the pathogen takes place. It was found that lesions and the pattern of the pathomorphological changes (destruction, necrosis and edema of the stroma of the villi, lysis of the cytoplasm, deformation of the enterocyte nuclei) in the small intestine of experimentally infected dogs depend on the development of the pathological process related not only to the changes in histoarchitectonics of the wall of the intestine, but also to tension of the histochemical statics, and obviously the dynamic of the cells (accumulation of the main and acidic proteins in enterocytes’ cytoplasm, hypersecretion of the mucus by goblet cells, decrease of Schiff iodine acid-positive substances in the enterocytes’ cytoplasm, formation of basophilous inclusion bodies), which leads to disorders in metabolic processes in the organism of infected dogs as a response to the virus infection. The examined dogs were found to have morphological changes in the small intestine similar to those in spontaneously infected animals. During the action of coronavirus, the contacts between the enterocytes become damaged, which leads to inhibition of the protective functions of the intestine. At the same time, the pathological process in the experimentally infected animals developed rapidly and had an acute course. Thus, coronavirus enteritis as a separate disease is practically unobserved in field conditions, which makes microscopic survey of the pathogenic impact of the latter on the organism of dogs impossible. Therefore, experimental mono-infection allows a detailed study to be conducted of pathomorphological changes of the initial place of the reproduction of the virus – the small intestine affected by coronavirus enteritis.