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This study evaluated the relationship between body size and digestive tract characteristics of the important predatory freshwater fish Hoplias malabaricus, which is widely distributed in South America. The allometric coefficients were calculated for the mass and standard length ( LS) relationships for two different LS groups: (1) between 20 and 100 mm (characterized as insectivores) and (2) >100 mm (characterized as piscivores). Differential growth measured from the allometric coefficient, b, between the insectivore ( b < 3) and the piscivore ( b > 3) groups was detected. Anterior intestine length and pyloric caeca zone length showed significant differences between groups. Two complementary hypotheses were developed to explain the differential growth: (1) H. malabaricus has a digestive tract adapted to a piscivorous diet, which is independent of its ontogenetic stage of development, and (2) the negative allometry observed in group 1 individuals agrees with a general behavioural strategy, allowing individuals to grow in LS during a shorter period of time. [ABSTRACT FROM AUTHOR]

Cellular demands for cholesterol are met by a balance between its biosynthesis in the endoplasmic reticulum (ER) and its uptake from lipoproteins. Cholesterol levels in intracellular membranes form a gradient maintained by a complex network of mechanisms including the control of the expression, compartmentalization and allosteric modulation of the enzymes that balance endogenous and exogenous sources of cholesterol. Low-density lipoproteins (LDLs) are internalized and delivered to lysosomal compartments to release their cholesterol content, which is then distributed within cellular membranes. High-density lipoproteins (HDLs), on the other hand, can transfer their cholesterol content directly into cellular membranes through the action of receptors such as the scavenger receptor B type 1 (SR-B1; gene SCARB1 ). We show here that SR-B1-mediated exogenous cholesterol internalization from HDL stimulates the formation of lipid-raft subdomains in the ER known as mitochondria-associated ER membranes (MAM), that, in turn, suppress de novo cholesterol biosynthesis machinery. We propose that MAM is a regulatory hub for cholesterol homeostasis that offers a novel dimension for understanding the intracellular regulation of this important lipid., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Genetic mutations causing primary mitochondrial disease (i.e those compromising oxidative phosphorylation [OxPhos]) resulting in reduced bioenergetic output display great variability in their clinical features, but the reason for this is unknown. We hypothesized that disruption of the communication between endoplasmic reticulum (ER) and mitochondria at mitochondria-associated ER membranes (MAM) might play a role in this variability. To test this, we assayed MAM function and ER-mitochondrial communication in OxPhos-deficient cells, including cybrids from patients with selected pathogenic mtDNA mutations. Our results show that each of the various mutations studied indeed altered MAM functions, but notably, each disorder presented with a different MAM "signature". We also found that mitochondrial membrane potential is a key driver of ER-mitochondrial connectivity. Moreover, our findings demonstrate that disruption in ER-mitochondrial communication has consequences for cell survivability that go well beyond that of reduced ATP output. The findings of a "MAM-OxPhos" axis, the role of mitochondrial membrane potential in controlling this process, and the contribution of MAM dysfunction to cell death, reveal a new relationship between mitochondria and the rest of the cell, as well as providing new insights into the diagnosis and treatment of these devastating disorders., (© 2024. The Author(s).)

Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer's disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-β as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes ("MAM" domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD., (© 2022. The Author(s).)

Ants, an ecologically successful and numerically dominant group of animals, play key ecological roles as soil engineers, predators, nutrient recyclers, and regulators of plant growth and reproduction in most terrestrial ecosystems. Further, ants are widely used as bioindicators of the ecological impact of land use. We gathered information of ant species in the Atlantic Forest of South America. The ATLANTIC ANTS data set, which is part of the ATLANTIC SERIES data papers, is a compilation of ant records from collections (18,713 records), unpublished data (29,651 records), and published sources (106,910 records; 1,059 references), including papers, theses, dissertations, and book chapters published from 1886 to 2020. In total, the data set contains 153,818 ant records from 7,636 study locations in the Atlantic Forest, representing 10 subfamilies, 99 genera, 1,114 ant species identified with updated taxonomic certainty, and 2,235 morphospecies codes. Our data set reflects the heterogeneity in ant records, which include ants sampled at the beginning of the taxonomic history of myrmecology (the 19th and 20th centuries) and more recent ant surveys designed to address specific questions in ecology and biology. The data set can be used by researchers to develop strategies to deal with different macroecological and region-wide questions, focusing on assemblages, species occurrences, and distribution patterns. Furthermore, the data can be used to assess the consequences of changes in land use in the Atlantic Forest on different ecological processes. No copyright restrictions apply to the use of this data set, but we request that authors cite this data paper when using these data in publications or teaching events., (© 2021 The Authors. Ecology © 2021 The Ecological Society of America.)

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

A technology capable of sequencing individual protein molecules would revolutionize our understanding of biological processes. Nanopore technology can analyze single heteropolymer molecules such as DNA by measuring the ionic current flowing through a single nanometer hole made in an electrically insulating membrane. This current is sensitive to the monomer sequence. However, proteins are remarkably complex and identifying a single residue change in a protein remains a challenge. In this work, I show that simple neural networks can be trained to recognize protein mutants. Although these networks are quickly and efficiently trained, their ability to generalize in an independent experiment is poor. Using a thermal annealing protocol on the nanopore sample, and examining many mutants with the same nanopore sensor are measures aimed at reducing training data variability which produce an increase in the generalizability of the trained neural network. Using this approach, we obtain a 100% correct assignment among 9 mutants in >50% of the experiments. Interestingly, the neural network performance, compared to a random guess, improves as more mutants are included in the dataset for discrimination. Engineered nanopores prepared with high homogeneity coupled with state-of-the-art analysis of the ionic current signals may enable single-molecule protein sequencing., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

In vivo proteins fold mainly as they emerge from the ribosome or as they emerge from a membrane translocon. Membrane translocation in particular poses technical challenges to the study of the associated protein folding processes. Recently we have developed a single-molecule methodology that allows the capture of a single protein molecule through a membrane translocon with biotinylated oligonucleotides covalently bound at its N- and C- terminus using streptavidin. The resulting rotaxane can be driven forwards and backwards changing the voltage polarity, and carefully planned experiments allow inference of the folding pathway. Here we will discuss the details of a simplified methodological approach., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nanopore technology enables the detection and analysis of single protein molecules. The technique measures the ionic current passing through a single pore inserted in an electrically insulating membrane. The translocation of the protein molecule through the pore causes a modulation of the ionic current. Analysis of the ionic current reveals the biophysics of co-translocational unfolding and may be used to infer the amino acid sequence and posttranslational modifications of the molecule.

The discovery of contact sites was a breakthrough in cell biology. We have learned that an organelle cannot function in isolation, and that many cellular functions depend on communication between two or more organelles. One such contact site results from the close apposition of the endoplasmic reticulum (ER) and mitochondria, known as mitochondria-associated ER membranes (MAMs). These intracellular lipid rafts serve as hubs for the regulation of cellular lipid and calcium homeostasis, and a growing body of evidence indicates that MAM domains modulate cellular function in both health and disease. Indeed, MAM dysfunction has been described as a key event in Alzheimer disease (AD) pathogenesis. Our most recent work shows that, by means of its affinity for cholesterol, APP-C99 accumulates in MAM domains of the ER and induces the uptake of extracellular cholesterol as well as its trafficking from the plasma membrane to the ER. As a result, MAM functionality becomes chronically upregulated while undergoing continual turnover. The goal of this review is to discuss the consequences of C99 elevation in AD, specifically the upregulation of cholesterol trafficking and MAM activity, which abrogate cellular lipid homeostasis and disrupt the lipid composition of cellular membranes. Overall, we present a novel framework for AD pathogenesis that can be linked to the many complex alterations that occur during disease progression, and that may open a door to new therapeutic strategies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol-rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER for cleavage by γ-secretase, acts as a lipid-sensing peptide that forms regulatory DRMs in the ER, called mitochondria-associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Understanding protein folding under conditions similar to those found in vivo remains challenging. Folding occurs mainly vectorially as a polypeptide emerges from the ribosome or from a membrane translocon. Protein folding during membrane translocation is particularly difficult to study. Here, we describe a single-molecule method to characterize the folded state of individual proteins after membrane translocation, by monitoring the ionic current passing through the pore. We tag both N and C termini of a model protein, thioredoxin, with biotinylated oligonucleotides. Under an electric potential, one of the oligonucleotides is pulled through a α-hemolysin nanopore driving the unfolding and translocation of the protein. We trap the protein in the nanopore as a rotaxane-like complex using streptavidin stoppers. The protein is subjected to cycles of unfolding-translocation-refolding switching the voltage polarity. We find that the refolding pathway after translocation is slower than in bulk solution due to the existence of kinetic traps.

Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 k B T, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.

Objective: Diabetes is characterized by pancreatic β-cell dedifferentiation. Dedifferentiating β cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the β-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear., Methods: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to β-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios., Results: The expression of Cyb5r3 is decreased in FoxO1-deficient β cells. Mice with β-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient β cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient β cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability., Conclusions: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate β-cell failure., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer's disease (AD). However, the reason for the association between APOE4 and AD remains unclear. While much of the research has focused on the ability of the apoE4 protein to increase the aggregation and decrease the clearance of Aβ, there is also an abundance of data showing that APOE4 negatively impacts many additional processes in the brain, including bioenergetics. In order to gain a more comprehensive understanding of APOE4's role in AD pathogenesis, we performed a transcriptomics analysis of APOE4 vs. APOE3 expression in the entorhinal cortex (EC) and primary visual cortex (PVC) of aged APOE mice. This study revealed EC-specific upregulation of genes related to oxidative phosphorylation (OxPhos). Follow-up analysis utilizing the Seahorse platform showed decreased mitochondrial respiration with age in the hippocampus and cortex of APOE4 vs. APOE3 mice, but not in the EC of these mice. Additional studies, as well as the original transcriptomics data, suggest that multiple bioenergetic pathways are differentially regulated by APOE4 expression in the EC of aged APOE mice in order to increase the mitochondrial coupling efficiency in this region. Given the importance of the EC as one of the first regions to be affected by AD pathology in humans, the observation that the EC is susceptible to differential bioenergetic regulation in response to a metabolic stressor such as APOE4 may point to a causative factor in the pathogenesis of AD.

Mitochondria are responsible for the generation of ATP by oxidative phosphorylation; however, these multifaceted organelles regulate many other key cellular functions as well, such as calcium homeostasis, apoptosis, and biosynthesis of steroid hormones, heme and phospholipids. In order to carry out these functions, mitochondria establish physical and functional connections with other organelles such as the plasma membrane, lipid droplets/vesicles, peroxisomes, endosomes, and the endoplasmic reticulum. Dysregulation of any of the aforementioned processes or inter-organelle contacts can lead to mitochondrial dysfunction and subsequent changes in oxygen consumption and ATP production. Seahorse technology has become a critical tool for quantification of mitochondrial oxygen consumption and can help differentiate primary mitochondrial disorders from disorders where alterations in mitochondrial metabolism are consequences of a prior, upstream insult. In this chapter, we describe the application of Seahorse technology for assaying mitochondrial respiration in whole cells, permeabilized cells and isolated mitochondria. We leave it to the researcher's discretion to determine which of these approaches will generate the most physiologically relevant data based on the model system and research question at hand., (© 2020 Elsevier Inc. All rights reserved.)

Inter-organelle communication is a rapidly-expanding field that has transformed our understanding of cell biology and pathology. Organelle-organelle contact sites can generate transient functional domains that act as enzymatic hubs involved in the regulation of cellular metabolism and intracellular signaling. One of these hubs is located in areas of the endoplasmic reticulum (ER) connected to mitochondria, called mitochondria-associated ER membranes (MAM). These MAM are transient lipid rafts intimately involved in cholesterol and phospholipid metabolism, calcium homeostasis, and mitochondrial function and dynamics. In addition, γ-secretase-mediated proteolysis of the amyloid precursor protein 99-aa C-terminal fragment (C99) to form amyloid β also occurs at the MAM. Our most recent data indicates that in Alzheimer's disease, increases in uncleaved C99 levels at the MAM provoke the upregulation of MAM-resident functions, resulting in the loss of lipid homeostasis, and mitochondrial dysfunction. Here, we discuss the relevance of these findings in the field, and the contribution of C99 and MAM dysfunction to Alzheimer's disease neuropathology., (© 2020 Elsevier Inc. All rights reserved.)

Charcot-Marie-Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)-mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER-mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT., (© The Author(s) 2019. Published by Oxford University Press.)

Thiazolidinediones (TZDs) are PPARγ agonists with potent insulin-sensitizing effects. However, their use has been curtailed by substantial adverse effects on weight, bone, heart, and hemodynamic balance. TZDs induce the deacetylation of PPARγ on K268 and K293 to cause the browning of white adipocytes. Here, we show that targeted PPARγ mutations resulting in constitutive deacetylation (K268R/K293R, 2KR) increased energy expenditure and protected from visceral adiposity and diet-induced obesity by augmenting brown remodeling of white adipose tissues. Strikingly, when 2KR mice were treated with rosiglitazone, they maintained the insulin-sensitizing, glucose-lowering response to TZDs, while displaying little, if any, adverse effects on fat deposition, bone density, fluid retention, and cardiac hypertrophy. Thus, deacetylation appears to fulfill the goal of dissociating the metabolic benefits of PPARγ activation from its adverse effects. Strategies to leverage PPARγ deacetylation may lead to the design of safer, more effective agonists of this nuclear receptor in the treatment of metabolic diseases.

Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction.

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the β-amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Protein interactions with specific DNA sequences are crucial in the control of gene expression and the regulation of replication. Single-molecule methods offer excellent capabilities to unravel the mechanism and kinetics of these interactions. Here, we develop a nanopore approach where a target DNA sequence is contained in a hairpin followed by a ssDNA. This system allows DNA-protein complexes to be distinguished from bare DNA molecules as they are pulled through a single nanopore detector, providing both equilibrium and kinetic information. We show that this approach can be used to test the inhibitory effect of small molecules on complex formation and their mechanisms of action. In a proof of concept, we use DNAs with different sequence patterns to probe the ability of the nanopore to distinguish the effects of an inhibitor in a complex mixture of target DNAs and proteins. We anticipate that the use of this technology with arrays of thousands of nanopores will contribute to the development of transcription factor binding inhibitors.

Protein unfolding and translocation through pores occurs during trafficking between organelles, protein degradation and bacterial toxin delivery. In vivo, co-translocational unfolding can be affected by the end of the polypeptide that is threaded into the pore first. Recently, we have shown that co-translocational unfolding can be followed in a model system at the single-molecule level, thereby unravelling molecular steps and their kinetics. Here, we show that the unfolding kinetics of the model substrate thioredoxin, when pulled through an α-haemolysin pore, differ markedly depending on whether the process is initiated from the C terminus or the N terminus. Further, when thioredoxin is pulled from the N terminus, the unfolding pathway bifurcates: some molecules finish unfolding quickly, while others finish ~100 times slower. Our findings have important implications for the understanding of biological unfolding mechanisms and in the application of nanopore technology for the detection of proteins and their modifications.

We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic current amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.

The stability of components of multiprotein complexes often relies on the presence of the functional complex. To assess structural dependence among the components of the R388 Type IV secretion system (T4SS), the steady-state level of several Trw proteins was determined in the absence of other Trw components. While several Trw proteins were affected by the lack of others, we found that the coupling protein TrwB is not affected by the absence of other T4SS components, nor did its absence alter significantly the levels of integral components of the complex, underscoring the independent role of the coupling protein on the T4SS architecture. The cytoplasmic ATPases TrwK (VirB4) and TrwD (VirB11) were affected by the absence of several core complex components, while the pilus component TrwJ (VirB5) required the presence of all other Trw proteins (except for TrwB) to be detectable. Overall, the results delineate a possible assembly pathway for the T4SS of R388. We have also tested structural complementation of TrwD (VirB11) and TrwJ (VirB5) by their homologues in the highly related Trw system of Bartonella tribocorum (Bt). The results reveal a correlation with the functional complementation data previously reported., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.

Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

Protein physicochemical properties must undergo complex changes during evolution, as a response to modifications in the organism environment, the result of the proteins taking up new roles or because of the need to cope with the evolution of molecular interacting partners. Recent work has emphasized the role of stability and stability-function trade-offs in these protein adaptation processes. In the present study, on the other hand, we report that combinations of a few conservative, high-frequency-of-fixation mutations in the thioredoxin molecule lead to largely independent changes in both stability and the diversity of catalytic mechanisms, as revealed by single-molecule atomic force spectroscopy. Furthermore, the changes found are evolutionarily significant, as they combine typically hyperthermophilic stability enhancements with modulations in function that span the ranges defined by the quite different catalytic patterns of thioredoxins from bacterial and eukaryotic origin. These results suggest that evolutionary protein adaptation may use, in some cases at least, the potential of conservative mutations to originate a multiplicity of evolutionarily allowed mutational paths leading to a variety of protein modulation patterns. In addition the results support the feasibility of using evolutionary information to achieve protein multi-feature optimization, an important biotechnological goal.

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwB Delta N70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.

We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Many enzymes possess, besides their native function, additional promiscuous activities. Proteins with several activities (multipurpose catalysts) may have a wide range of biotechnological and biomedical applications. Natural promiscuity, however, appears to be of limited scope in this context, because the latent (promiscuous) function is often related to the evolved one (sharing the active site and even the chemical mechanism) and its enhancement upon suitable mutations usually brings about a decrease in the native activity. Here we explore the use of computational protein design to overcome these limitations. The high-plasticity positions close to the original ("native") active-site are the most promising candidates for mutations that create a second active-site associated to a new function. To avoid compromising protein folding and native activity, we propose a minimal-perturbation approach based on the combinatorial optimization of, both the de novo catalytic activity and the folding free-energy: essentially, we construct the Pareto Set of optimal stability/promiscuous-function solutions. We validate our approach by introducing a promiscuous esterase activity in E. coli thioredoxin on the basis of mutations at positions close to the native-active-site disulfide-bridge. Native oxidoreductase activity is not compromised and it is, in fact, found to be 1.5-fold enhanced, as determined by an insulin-reduction assay. This work provides general guidelines as to how computational design can be used to expand the scope and applications of protein promiscuity. From a more general viewpoint, it illustrates the potential of multi-objective optimization as the computational analogue of multi-feature natural selection., (Copyright 2009 Elsevier B.V. All rights reserved.)

Recent work has shown that proteins can tolerate hydrophobic-to-ionizable-residue mutations. Here, we provide experimental evidence that the essential properties (pK value, protonation state, local dynamics) of buried ionizable groups in proteins can be efficiently modulated through the rational design of the surface charge distribution, thus paving the way for the protein engineering exploitation of charge burial.

A large collection of structural snapshots along a full catalytic cycle of Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using a combination of theoretical methods. Molecular models were built starting from the available X-ray crystallographic structures of dimeric wild-type TrxR in the flavin-oxidizing conformation and a C135S TrxR mutant enzyme in a flavin-reducing conformation "trapped" by a cross-link between Cys138 of TrxR and Cys32 of C35S mutant thioredoxin (Trx). The transition between these two extreme states, which is shown to be reproduced in a normal mode analysis, as well as natural cofactor binding and dissociation, were simulated for the wild-type species using unrestrained and targeted molecular dynamics following docking of oxidized Trx to reduced TrxR. The whole set of simulations provides a comprehensive structural framework for understanding the mechanism of disulfide reduction in atomic detail and identifying the most likely intermediates that facilitate entry of NADPH and exit of NADP(+). The crucial role assigned to Arg73 and Lys36 of Trx in substrate binding and complex stabilization was ascertained when R73G, R73D, and K36A site-directed mutants of Trx were shown to be impaired to different extents in their ability to be reduced by TrxR. On the basis of previous findings and the results reported herein, E. coli TrxR appears as a beautifully engineered molecular machine that is capable of synchronizing cofactor capture and ejection with substrate binding and redox activity through an interdomain twisting motion., ((c) 2009 Wiley-Liss, Inc.)

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1-43)) and Ser(19)-phosphorylated (THp-(1-43)) states by surface plasmon resonance. THp-(1-43) showed high affinity for 14-3-3 proteins (K(d) approximately 0.5 microM for 14-3-3gamma and -zeta and 7 microM for 14-3-3eta). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S(0.5) = 25-58 microM (TH-(1-43)) and S(0.5) = 135-475 microM (THp-(1-43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3gamma showed a preferential binding to membranes, compared with 14-3-3zeta, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3gamma for negatively charged membranes (S(0.5) = 1-9 microM) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser(19)-phosphorylated TH, 14-3-3gamma, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of L-DOPA and dopamine synthesis.