Ismael Mingarro | University of Valencia, Spain (original) (raw)

Papers by Ismael Mingarro

Journal of Molecular Biology, Mar 1, 2022

Salt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an impor... more Salt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. It is possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we show that potentially salt-bridge forming pairs are statistically over-represented in TM-helices. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and whole cell systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute ∼0.5 kcal/mol to the apparent free energy of membrane insertion (ΔGapp). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.

Virology Journal, May 24, 2023

to reduce the morbidity and mortality associated with COVID-19 disease. The cellular membrane is ... more to reduce the morbidity and mortality associated with COVID-19 disease. The cellular membrane is the first barrier the virus encounters when infecting a new cell. At some point during entry, enveloped viruses must fuse the cellular and viral membranes. In the case of SARS-CoV-2, the highly glycosylated spike (S) protein is responsible for gaining entry to the host cells [3]. The S protein, a class I trimeric fusion protein [4], is translated in a non-active form (S 0). Proteolytic activation of S 0 by host proteases (i.e. TMPRSS2) produces the mature pre-fusion S protein incorporated in the virions [5]. The infectious process starts with the binding of S protein to the angiotensinconverting enzyme 2 (ACE2) at the cell surface [2, 6]. Binding to ACE2 triggers a conformational change, resulting in the exposure of the S protein fusion peptide, which will interact with the host membrane and initiate the fusion of the viral and cellular membranes. Facilitating any of the multiple steps in this complex process would speed up and increase viral production, expediting Virology Journal

Biochimica Et Biophysica Acta - Biomembranes, Apr 1, 2006

Part of the content of the manuscript has been presented as an invited talk to the 'Third Europea... more Part of the content of the manuscript has been presented as an invited talk to the 'Third European contest for young scientists on Challenging Proteins. Amersham Bioscience', Paris 2005, where it has been awarded by an international jury.

Biochemical and Biophysical Research Communications, Jul 1, 2015

Transient Receptor Potential (TRP) channels are related to adaptation to the environment and soma... more Transient Receptor Potential (TRP) channels are related to adaptation to the environment and somatosensation. The transient receptor potential vanilloid (TRPV) subfamily includes six closely evolutionary related ion channels sharing the same domain organization and tetrameric arrangement in the membrane. In this study we have characterized biochemically TRPV2 channel membrane protein folding and transmembrane (TM) architecture. Deleting the first N-terminal 74 residues preceding the ankyrin repeat domain (ARD) show a key role for this region in targeting the protein to the membrane. We have demonstrated the co-translational insertion of the membrane-embedded region of the TRPV2 and its disposition in biological membranes, identifying that TM1-TM4 and TM5-TM6 regions can assemble as independent folding domains. The ARD is not required for TM domain insertion in the membrane. The folding features observed for TRPV2 may be conserved and shared among other TRP channels outside the TRPV subfamily.

Protein Science, 2000

The monomer-dimer equilibrium of the glycophorin A~GpA! transmembrane~TM! fragment has been used ... more The monomer-dimer equilibrium of the glycophorin A~GpA! transmembrane~TM! fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization.

Protein Science, Dec 10, 2010

N-glycosylation is the most common and versatile protein modification. In eukaryotic cells, this ... more N-glycosylation is the most common and versatile protein modification. In eukaryotic cells, this modification is catalyzed cotranslationally by the enzyme oligosaccharyltransferase, which targets the b-amide of the asparagine in an Asn-Xaa-Ser/Thr consensus sequon (where Xaa is any amino acid but proline) in nascent proteins as they enter the endoplasmic reticulum. Because modification of the glycosylation acceptor site on membrane proteins occurs in a compartment-specific manner, the presence of glycosylation is used to indicate membrane protein topology. Moreover, glycosylation sites can be added to gain topological information. In this study, we explored the determinants of N-glycosylation with the in vitro transcription/translation of a truncated model protein in the presence of microsomes and surveyed 25,488 glycoproteins, of which 2,533 glycosylation sites had been experimentally validated. We found that glycosylation efficiency was dependent on both the distance to the C-terminus and the nature of the amino acid that preceded the consensus sequon. These findings establish a broadly applicable method for membrane protein tagging in topological studies.

Proceedings of the National Academy of Sciences of the United States of America, Dec 27, 2016

Biochemistry, Jul 31, 2004

Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria... more Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapoptotic Bcl-x(L) and proapoptotic Bax and Bid, that correspond to defined alpha helices in the structure of their soluble forms. According to prediction methods, there are only two putative TM fragments in Bcl-x(L) and Bax (the C-terminal alpha helix and alpha-helix 5) and one in activated tBid (alpha-helix 6). The rest of their sequence, including the second helix of the pore-forming domain, displays only weak hydrophobic peaks, which are below the prediction threshold. Subsequent analysis by glycosylation mapping of single alpha-helix segments in a model chimeric system confirms the above predictions and allows finding an extra TM fragment made of helix alpha1 of Bax. Surprisingly, the amphipathic helices alpha6 of Bcl-x(L) and Bax and alpha7 of Bid do insert in membranes only as part of the alpha5-alpha6 (Bcl-x(L) and Bax) or alpha6-alpha7 (Bid) hairpins but not when assayed individually. This behavior suggests a synergistic insertion and folding of the two helices of the hairpin that could be due to charge complementarity and additional stability provided by turn-inducing residues present at the interhelical region. Although these data come from chimeric systems, they show direct potentiality for acquiring a membrane inserted state. Thus, the above fragments should be considered for the definition of plausible models of the active, membrane-bound species of Bcl-2 proteins.

ChemBioChem, Jul 8, 2005

Plant viral movement proteins bind to RNA and participate in the intra- and intercellular movemen... more Plant viral movement proteins bind to RNA and participate in the intra- and intercellular movement of the RNAs from plant viruses. However, the role and magnitude of the conformational changes associated with the formation of RNA-protein complexes are not yet defined. Here we describe studies on the relevance of a preexisting nascent alpha-helix at the C terminus of the RNA-binding domain of p7, a movement protein from carnation mottle virus, to RNA binding. Synthetic peptide analogues and single amino acid mutation at the RNA-binding domain of recombinant p7 protein were used to correlate the transient structural order in aqueous solution with RNA-binding potential.

Biochemistry, Apr 19, 1994

The behavior of porcine pancreatic phospholipase A2 (ppPLA2) in monophasic low-water media has be... more The behavior of porcine pancreatic phospholipase A2 (ppPLA2) in monophasic low-water media has been explored, for the first time, in a systematic manner. It has been investigated how a number of variables can modulate both acylating and deacylating activities of the enzyme, and several interesting, unexpected results are presented. Among the most relevant, when placing ppPLA2 in the water-restricted environment, are the following: (i) it displays a remarkable alteration of its specificity toward the substrate polar head relative to all-water medium; (ii) it is quite severely inhibited by lysophosphatidylcholine (LPC), which has important implications, particularly concerning its acylation activity; and (iii) it exquisitely discriminates between saturated and unsaturated long-chain fatty acids when esterifying them with LPC. Finally, it is also illustrated how these results can be exploited to optimize the catalytic performance of the enzyme in nonaqueous medium and obtain a nearly 30-fold increase in the yield of phosphatidylcholine synthesis with respect to previously reported data.

FEBS Letters, Feb 16, 2005

The principles that govern the folding and packing of membrane proteins are still not completely ... more The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimerisation motif that mediates transmembrane (TM) helix association, one of the best-suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Further, we have used this minimised GpA motif to assess the influence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helixhelix association and/or dissociation between TM fragments.

Journal of Biological Chemistry, Mar 1, 2000

We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with s... more We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic Nterminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the Ntail remains mostly nontranslocated and the majority of the molecules adopt an "inverted" topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-toN terminal direction.

Journal of Molecular Biology, Oct 1, 1997

The unique ability of the glycophorin A transmembrane helix to dimerize in SDS has previously bee... more The unique ability of the glycophorin A transmembrane helix to dimerize in SDS has previously been exploited in studies of the sequence speci-®city of helix-helix packing in a micellar environment. Here, we have made different insertion mutants in the critical helix-helix interface segment, and ®nd that ef®cient dimerization can be mediated by a wider range of sequence motifs than suggested by the earlier studies. We also show that certain mutants that are unable to dimerize can nevertheless form relatively high amounts of tetramers, and that speci®c tetramerization can be induced by duplication of the critical interface motif on the lipid-exposed side of the transmembrane helix.

Proceedings of the National Academy of Sciences of the United States of America, Oct 22, 2020

PLOS ONE, Sep 12, 2012

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic a... more The vast majority of membrane proteins are anchored to biological membranes through hydrophobic a-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.

Journal of Medicinal Chemistry, Feb 1, 2005

Two peptoids that neutralize the Gram-negative lipopolysaccharide (LPS) were identified from the ... more Two peptoids that neutralize the Gram-negative lipopolysaccharide (LPS) were identified from the screening of a positional scanning library. The evaluation of the in vivo activity of these compounds in an endoxemia murine model is also reported. These peptoids did not neutralize lipid A, i.e., the hydrophobic toxic component of LPS. This fact suggests that they do not have access to the micellar core and that they should bind to the hydrophilic carbohydrate portion of LPS.

Journal of Molecular Biology, 2004

Current Medicinal Chemistry, Feb 1, 2008

Pulmonary surfactant is a lipid-protein complex that coats the interior of the alveoli and enable... more Pulmonary surfactant is a lipid-protein complex that coats the interior of the alveoli and enables the lungs to function properly. Upon its synthesis, lung surfactant adsorbs at the interface between the air and the hypophase, a capillary aqueous layer covering the alveoli. By lowering and modulating surface tension during breathing, lung surfactant reduces respiratory work of expansion, and stabilises alveoli against collapse during expiration. Pulmonary surfactant deficiency, or dysfunction, contributes to several respiratory pathologies, such as infant respiratory distress syndrome (IRDS) in premature neonates, and acute respiratory distress syndrome (ARDS) in children and adults. The main clinical exogenous surfactants currently in use to treat some of these pathologies are essentially organic extracts obtained from animal lungs. Although very efficient, natural surfactants bear serious defects: i) they could vary in composition from batch to batch; ii) their production involves relatively high costs, and sources are limited; and iii) they carry a potential risk of transmission of animal infectious agents and the possibility of immunological reaction. All these caveats justify the necessity for a highly controlled synthetic material. In the present review the efforts aimed at new surfactant development, including the modification of existing exogenous surfactants by adding molecules that can enhance their activity, and the progress achieved in the production of completely new preparations, are discussed.