Renal control of disease tolerance to malaria Ramos, Susana; Carlos, Ana Rita; Sundaram, Balamurugan ...
Proceedings of the National Academy of Sciences - PNAS,
03/2019, Letnik:
116, Številka:
12
Journal Article
Recenzirano
Odprti dostop
Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill ...Plasmodium. However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.
'Genomic design' refers to the structural organization of gene sequences. Recently, the role of intron sequences for gene regulation is being better understood. Further, introns possess high rates of ...polymorphism that are considered as the major source for speciation. In molecular breeding, the length of gene-specific introns is recognized as a tool to discriminate genotypes with diverse traits of agronomic interest. 'Economy selection' and 'time-economy selection' have been proposed as models for explaining why highly expressed genes typically contain small introns. However, in contrast to these theories, plant-specific selection reveals that highly expressed genes contain introns that are large. In the presented research, 'wet'Aox gene identification from grapevine is advanced by a bioinformatics approach to study the species-specific organization of Aox gene structures in relation to available expressed sequence tag (EST) data. Two Aox1 and one Aox2 gene sequences have been identified in Vitis vinifera using grapevine cultivars from Portugal and Germany. Searching the complete genome sequence data of two grapevine cultivars confirmed that V. vinifera alternative oxidase (Aox) is encoded by a small multigene family composed of Aox1a, Aox1b and Aox2. An analysis of EST distribution revealed high expression of the VvAox2 gene. A relationship between the atypical long primary transcript of VvAox2 (in comparison to other plant Aox genes) and its expression level is suggested. V. vinifera Aox genes contain four exons interrupted by three introns except for Aox1a which contains an additional intron in the 3'-UTR. The lengths of primary Aox transcripts were estimated for each gene in two V. vinifera varieties: PN40024 and Pinot Noir. In both varieties, Aox1a and Aox1b contained small introns that corresponded to primary transcript lengths ranging from 1501 to 1810 bp. The Aox2 of PN40024 (12 329 bp) was longer than that from Pinot Noir (7279 bp) because of selection against a transposable-element insertion that is 5028 bp in size. An EST database basic local alignment search tool (BLAST) search of GenBank revealed the following ESTs percentages for each gene: Aox1a (26.2%), Aox1b (11.9%) and Aox2 (61.9%). Aox1a was expressed in fruits and roots, Aox1b expression was confined to flowers and Aox2 was ubiquitously expressed. These data for V. vinifera show that atypically long Aox intron lengths are related to high levels of gene expression. Furthermore, it is shown for the first time that two grapevine cultivars can be distinguished by Aox intron length polymorphism.
Heme, iron (Fe) protoporphyrin IX, functions as a prosthetic group in a range of hemoproteins essential to support life under aerobic conditions. The Fe contained within the prosthetic heme groups of ...these hemoproteins can catalyze the production of reactive oxygen species. Presumably for this reason, heme must be sequestered within those hemoproteins, thereby shielding the reactivity of its Fe-heme. However, under pathologic conditions associated with oxidative stress, some hemoproteins can release their prosthetic heme groups. While this heme is not necessarily damaging per se, it becomes highly cytotoxic in the presence of a range of inflammatory mediators such as tumor necrosis factor. This can lead to tissue damage and, as such, exacerbate the pathologic outcome of several immune-mediated inflammatory conditions. Presumably, targeting "free heme" may be used as a therapeutic intervention against these diseases.
Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, ...macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro‐oxidant manner and regulates cellular metabolism while exerting pro‐inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme‐specific single domain antibodies (sdAbs) that together with a cellular‐based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10−7 m and that 2–8% (~ 2–5 μm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme‐binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10−7 m. The heme‐specific sdAbs neutralize the pro‐oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme‐specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme‐specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme.
During hemolysis, extracellular hemoglobin can release its heme groups upon oxidation. The resulting labile heme is exchanged between different plasma acceptor proteins, macromolecules and low molecular weight ligands. Arrows indicate heme transfer from lower to higher affinity acceptors. Interactions are not at scale nor accurate according to crystal structures. Heme affinities are indicative for human plasma acceptors.
The fibroblast growth factor receptor 4 (FGFR4) is overexpressed in rhabdomyosarcoma (RMS) and represents a promising target for treatments based on specific and efficient antibodies. Despite ...progress, there is an urgent need for targeted treatment options to improve survival rates, and to limit long-term side effects. From phage display libraries we selected FGFR4-specific single-domain antibodies (sdAb) binding to recombinant FGFR4 and validated them by flow cytometry, surface plasmon resonance, and fluorescence microscopy. The specificity of the selected sdAb was verified on FGFR4-wild type and FGFR4-knock out cells. FGFR4-sdAb were used to decorate vincristine-loaded liposomes and to generate chimeric antigen receptor (CAR) T cells. First, incubation of RMS cells with FGFR4-sdAb revealed that FGFR4-sdAb can block FGF19-FGFR4 signaling via the MAPK pathway and could therefore serve as therapeutics for FGFR4-dependent cancers. Second, FGFR4-targeted vincristine-loaded liposomes bound specifically to RMS cells and were internalized by the receptor, demonstrating the potential for active drug delivery to the tumor. Third, FGFR4-CAR T cells, generated with one sdAb candidate, demonstrated strong and specific cytotoxicity against FGFR4 expressing RMS cells. We selected novel FGFR4-sdAb with high specificity and nano- to picomolar affinities for FGFR4 which have the potential to enable multiple FGFR4-targeted cancer therapy approaches.
Severe presentations of malaria emerge as
parasites invade and lyse red blood cells (RBC), producing extracellular hemoglobin (HB), from which labile heme is released. Here, we tested whether ...scavenging of extracellular HB and/or labile heme, by haptoglobin (HP) and/or hemopexin (HPX), respectively, counter the pathogenesis of severe presentations of malaria. We found that circulating labile heme is an independent risk factor for cerebral and non-cerebral presentations of severe
malaria in children. Labile heme was negatively correlated with circulating HP and HPX, which were, however, not risk factors for severe
malaria. Genetic
and/or
deletion in mice led to labile heme accumulation in plasma and kidneys, upon
This was associated with higher incidence of mortality and acute kidney injury (AKI) in ageing but not adult
-infected mice, and was corroborated by an inverse correlation between heme and HPX with serological markers of AKI in
malaria. In conclusion, HP and HPX act in an age-dependent manner to prevent the pathogenesis of severe presentation of malaria in mice and presumably in humans.
Unlocking cell secretion capacity is of paramount interest for the pharmaceutical industry focused on biologics. Here, we leveraged retention using a selective hook (RUSH) system for the ...identification of human osteosarcoma U2OS cell secretion modulators, through automated, high-throughput screening of small compound libraries. We created a U2OS cell line which co-expresses a variant of streptavidin addressed to the lumen-facing membrane of the endoplasmic reticulum (ER) and a recombinant anti-PD-L1 antibody. The heavy chain of the antibody was modified at its C-terminus, to which a furin cleavage site, a green fluorescent protein (GFP), and a streptavidin binding peptide (SBP) were added. We show that the U2OS cell line stably expresses the streptavidin hook and the recombinant antibody bait, which is retained in the ER through the streptavidin-SBP interaction. We further document that the addition of biotin to the culture medium triggers the antibody release from the ER, its trafficking through the Golgi where the GFP-SBP moiety is clipped off, and eventually its release in the extra cellular space, with specific antigen-binding properties. The use of this clone in screening campaigns led to the identification of lycorine as a secretion enhancer, and nigericin and tyrphostin AG-879 as secretion inhibitors. Altogether, our data support the utility of this approach for the identification of agents that could be used to improve recombinant production yields and also for a better understanding of the regulatory mechanism at work in the conventional secretion pathway.
The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these ...proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (K d ≈ 0.04−0.08 μM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (K d ≈ 0.3 μM for GSU0582 versus 17 μM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.
To reach the lysosome, lysosomal membrane proteins (LMPs) are translocated in the endoplasmic reticulum after synthesis and then transported to the Golgi apparatus. The existence of a direct ...transport from the Golgi apparatus to the endosomes but also of an indirect route through the plasma membrane has been described. Clathrin adaptor binding motifs contained in the cytosolic tail of LMPs have been described as key players in their intracellular trafficking. Here we used the RUSH assay to synchronize the biosynthetic transport of multiple LMPs. After exiting the Golgi apparatus, RUSH-synchronized LAMP1 was addressed to the cell surface both after overexpression or at endogenous level. Its YXXΦ motif was not involved in the transport from the Golgi apparatus to the plasma membrane but in its endocytosis. LAMP1 and LIMP2 were sorted from each other after reaching the Golgi apparatus. LIMP2 was incorporated in punctate structures for export from the Golgi apparatus from which LAMP1 is excluded. LIMP2-containing post-Golgi transport intermediates did not rely neither on its adaptor binding signal nor on its C-terminal cytoplasmic domain.
Hemo, i.e. ferro (Fe) contido na porfirina IX, funciona como grupo prostético numa variedade de hemoproteins que exercem funções biológicas vitais e portanto, essenciais para sustentar a vida. Hemo é ...uma molecula extremamente reactiva, podendo participar em reacções redox e presumivelmente por esse motivo deve ser sequestrado no interior de um bolso hémico nas hemoproteínas, para controlo da sua reactividade. Contudo, sob condições de stress biológico, as hemoproteínas podem libertar o seu grupo prostético formando “hemo livre” que liga-se fracamente às proteínas, ou outras moléculas adquirindo presumivelmente actividade redox descontrolada. Para além disso, um crescente número de evidências suporta a noção de que o “hemo livre” pode comportar-se como um agente vasoativo, pro - inflamatório e citotóxico quando libertado de um subconjunto de hemoproteínas como por exemplo, a extracelular hemoglobina gerada durante condições hemolíticas. Assim sendo, eventualmente o “hemo livre” contribui para a patogénese de doenças inflamatórias imuno-mediadas associadas com hemólise. As actuais limitações das abordagens metodológicas que possibilitem a análise quantitativa e qualitativa do “hemo livre” impedem a determinação inequívoca dos seus efeitos patológicos, tal como o seu eventual tratamento terapêutico. Para superar estas limitações, usamos a technologia de exibição de fagos para gerar um painel de anticorpos de domínio único (‘single domain antibodies’, sdAbs), nomeadamente cadeia variável leve (VL), que reconhecem específicamente “hemo livre” após libertação das respectivas hemoproteínas.O sdAb 2H10 foi selecionado, uma vez que é o mais específico para o “hemo livre” e a sua ligação ao hemo foi caracterizada usando as várias técnicas espectroscopicas (absorção no UV-visível, Raman ressonante, Dicroísmo circular). Estas técnicas demonstraram a presença de um hemo férrico seis coordenado de baixo ‘spin’, com uma cisteína como quinto ligando e, possívelmente uma lisina ou tirosina como o sexto ligado. Para além disso, a estructura secondária do sdAb, composta principalmente por folhas β antiparalelas, sofre alterações conformacionais para acomodar o hemo. Foi também demonstrado que estes sdAbs podem ser usados para detectar “hemo livre” em células humanas atravês de técnicas de imunofluorescência ou citometria de fluxo, tal como no plasma utilizando um ensaio ‘enzyme linked immunoabsorbant assay’ (ELISA), desenvolvido a partir dos sdAbs.Para verificar a existência do “hemo livre” no plasma, utilizámos um modelo experimental previamente estabelecido usando a fenilhidrazina (PHZ) para induzir hemólise aguda em murganhos. Neste ensaio o “hemo livre” não é detectado no plasma, mesmo quando os níveis de hemo total e de absorção celular do hemo aumentam, enquanto que a capacidade do plasma para ligar o hemo diminui transitoriamente. Eventualmente, a elevada capacidade de tampão do plasma é assegurada por proteínas, ou outras moléculas biológicas, proporcionando um mecanismo preciso e regulador para a manutenção da homeostase sistémica do hemo. Demonstrámos ainda que a ligação de sdAb 2H10 ao hemo reduz a sua actividade redox, ainda mais eficientemente do que o seu eliminador plasmático, albumina, uma acção que possivelmente possibilitará a inhibição dos efeitos patológicos do hemo.Em resumo, nós demonstramos que estes sdAbs podem ser usados para ultrapassar as actuais limitações relativas à análise quantitativa e qualitativa do “heme livre” em condições hemolíticas, ou noutras condições biológicas associadas presumivelmente com a libertação do hemo das hemoproteinas. Propõe-se a utilização dos sdAbs para fins terapêuticos, nomeadamente do sdAb 2H10, na captação do “hemo livre”, tal como na modulação da sua actividade redox em patologias associadas com hemólise.
PDF
