Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-perforin

The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain

LACTOFERIN IN THE PROBLEM OF ANTI-INFECTIOUS PROTECTION OF BABIES IN THEIR FIRST YEAR OF LIVING

The author  summarizes  the  results  of research  of the  antibacterial,  antiviral  and  antifungal  properties  of multifunctional  human protein  — lactoferrin,  in order  to determine  the prospects  for its use in the prevention  and  treatment  of infectious  diseases  of children in their first year of life. The mechanisms of anti-infectious effect of this protein with breastfed children have been described. Basic differences between human lactoferrin and cattle lactoferrin have been shown. Biotechnology of obtaining recombinant human lactoferrin from the milk of genetically engineered dairy animals (goat-producers) has been described. According to the studies, both by physical and chemical parameters and biological activity, human lactoferrin, obtained from milk-producing  goats, corresponds to its natural counterpart

Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

A Draft of the Human Septin Interactome

Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the ""group rule"", i.e. members of the same group (e. g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.Fundao de Amparo a Pesquisa do Estado Sao Paulo (Fapesp)CAPES: Coordenao de Aperfeioamento de Pessoal de Navel SuperiorConselho Nacional de Pesquisa e Desenvolvimento (CNPq)Laboratorio Nacional de Biociencias-Centro Nacional de Pesquisa em Energia e Materais (LNBio-CNPEM

Best practices for managing large CryoEM facilities

Microscopic imaging and technolog

ВОЗМОЖНОСТИ ПРИМЕНЕНИЯ ЛАКТОФЕРРИНА У ДЕТЕЙ ПЕРВОГО ГОДА ЖИЗНИ

The author  summarizes  the  results  of research  of the  antibacterial,  antiviral  and  antifungal  properties  of multifunctional  human protein  — lactoferrin,  in order  to determine  the prospects  for its use in the prevention  and  treatment  of infectious  diseases  of children in their first year of life. The mechanisms of anti-infectious effect of this protein with breastfed children have been described. Basic differences between human lactoferrin and cattle lactoferrin have been shown. Biotechnology of obtaining recombinant human lactoferrin from the milk of genetically engineered dairy animals (goat-producers) has been described. According to the studies, both by physical and chemical parameters and biological activity, human lactoferrin, obtained from milk-producing  goats, corresponds to its natural counterpart.В статье обобщены результаты исследований, посвященных изучению антибактериальных, противовирусных и противогрибковых свойств многофункционального белка человека — лактоферрина, для определения перспектив его применения с целью профилактики и лечения инфекционной патологии у детей первого года жизни. Описаны механизмы противоинфекционного действия этого белка у детей, находящихся на грудном вскармливании. Показаны основные различия между лактоферрином человека и лактоферрином крупного рогатого скота. Описана биотехнология получения рекомбинантного лактоферрина человека из молока генно-инженерных молочных животных (коз-продуцентов). Согласно проведенным исследованиям, как по физико-химическим параметрам, так и по биологической активности лактоферрин  человека, выделенный из молока коз-продуцентов, соответствует его природному аналогу

БИФИДОГЕННЫЕ СВОЙСТВА БИОТЕХНОЛОГИЧЕСКОГО АНАЛОГА ЛАКТОФЕРРИНА ЧЕЛОВЕКА

Background:  Recent research shows that the growth and development of the gastrointestinal  tract of children fed by breast milk is more intense than that of the formula fed, since the human lactoferrin contained in the breast milk is a factor that stimulates cell growth. Therefore, the possibility of using exogenous lactoferrin will be of great importance in the nutrition of infants.Objektive: To study the bifidogenic properties of the biotechnological analogue of human lactoferrin. Methods: Kinetics of growth and CFU titer of bifidobacterial culture in the presence of a biotechnological analogue of human lactoferrin (0,05–5 mg /ml) was determined.Results: It has been shown that different concentrations of the protein can have both a stimulating (for B. bifidum and B. infantis) and inhibitory (for B. longum) effect on the growth of bifidobacteria, which is due to the affinity of lactoferrin binding to them. It seems important to further study the stimulating effect of this protein on the growth of lactobacilli in the intestine of the child.Conclusion:  Due to bifidogenic and high bactericidal action, lactoferrin can be effective in feeding newborns.Обоснование.   Новейшие исследования  показывают,  что  рост  и развитие  желудочно-кишечного  тракта  детей, вскармливаемых  материнским молоком,  происходит  интенсивнее,  чем у вскармливаемых  молочными смесями, поскольку содержащийся в нем лактоферрин человека является фактором, стимулирующим клеточный рост. Именно поэтому возможность  использования экзогенного лактоферрина  будет иметь большую значимость в питании грудных детей.Цель  исследования — изучить бифидогенные  свойства биотехнологического аналога  лактоферрина человека.Методы. Определялась кинетика роста и КОЕ-титр культивирования бифидобактерий в присутствии биотехнологического  аналога лактоферрина человека.Результаты. Показано, что различные концентрации данного белка (0,05–5 мг/мл) могут оказывать как стимулирующее (для Bifidobacterium bifidum и Bifidobacterium infantis), так и ингибирующее (для Bifidobacterium longum) действие в отношении роста бифидобактерий,  что обусловлено аффинностью  связывания с ними лактоферрина. Представляется  важным дальнейшее изучение стимулирующего эффекта  этого белка на рост лактобацилл в кишечнике ребенка.Заключение. Благодаря  бифидогенному и выраженному бактерицидному действию лактоферрин может быть использован в лечебном питании новорожденных

Single-molecule kinetics of pore assembly by the membrane attack complex

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions

Intrinsically disordered protein threads through the bacterial outer-membrane Porin OmpF

Porins are β-barrel outer-membrane proteins through which small solutes and metabolites diffuse that are also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at the surface of Escherichia coli that incorporates the trimeric porin OmpF. Formation of the translocon involved ColE9’s unstructured N-terminal domain threading in opposite directions through two OmpF subunits, capturing its target TolB on the other side of the membrane in a fixed orientation that triggers colicin import. Thus, an intrinsically disordered protein can tunnel through the narrow pores of an oligomeric porin to deliver an epitope signal to the cell to initiate cell death