Complex Folding Research Articles

Skin and Nipple-Areola Complex Sparing Mastectomy in Breast Cancer Patients

Skin and nipple-areola complex sparing mastectomy (SNSM) and primary reconstruction have been popular for breast cancer treatment in the last decade. An advantage of the SNSM technique is the removal of all breast tissue as a radical surgical procedure while preserving native breast integrity, nipple-areola complex (NAC), and submammary fold. This retrospective 15-year clinical study analyzes medical records from our breast surgery database collected at our department between 1997 and 2012. A total number of 3757 patients were treated for breast cancer; 411 (10.9%) patients had a skin-sparing mastectomy with the median (range) length follow-up of 63 months. This is the longest follow-up for SNSM in breast cancer patients; 3.7% of patients who underwent SNSM developed disease local recurrence, whereas occult NAC involvement with cancer occurred in 7.7% and local recurrence in the NAC in 1.2%. Partial necrosis of the NAC developed in 9.4% and total necrosis in 0.7% of operated breasts. All disease recurrences occurred in the first 10 years of the follow-up period. Local recurrence developed as first recurrence event has longer median cancer-specific survival time of 70 months than those with only distant metastases with 50 months and locoregional plus distant metastases with 35.5 months. The "Omega" pattern incision combines an oncological radical procedure with a lower incidence of skin flap necrosis. Patients reconstructed with autologous tissue were the group most satisfied. SNSM is an oncological safe procedure for breast cancer treatment with low recurrence in properly selected patients.

Reconstruction and morphometric analysis of the nasal airway of the white-tailed deer (Odocoileus virginianus) and implications regarding respiratory and olfactory airflow.

Compared with other mammals (e.g., primates, rodents, and carnivores), the form and function of the ungulate nasal fossa, in particular the ethmoidal region, has been largely unexplored. Hence, the nasal anatomy of the largest prey species remains far less understood than that of their predators, rendering comparisons and evolutionary context unclear. Of the previous studies of nasal anatomy, none have investigated the detailed anatomy and functional morphology of the white-tailed deer (Odocoileus virginianus), a species that is ubiquitous throughout North and Central America and in northern regions of South America. Here, nasal form and function is quantitatively investigated in an adult white-tailed deer using high-resolution magnetic resonance imaging, combined with anatomical reconstruction and morphometric analysis techniques. The cross-sectional anatomy of the airway is shown and a three-dimensional anatomical model of the convoluted nasal fossa is reconstructed from the image data. A detailed morphometric analysis is presented that includes quantitative distributions of airway size and shape (e.g., airway perimeter, cross-sectional area, surface area) and the functional implications of these data regarding respiratory and olfactory airflow are investigated. The white-tailed deer is shown to possess a long, double scroll maxilloturbinal that occupies approximately half of the length of the nasal fossa and provides a large surface area for respiratory heat and moisture exchange. The ethmoidal region contains a convoluted arrangement of folded ethmoturbinals that appear to be morphologically distinct from the single and double scroll ethmoturbinals found in most other non-primates. This complex folding provides a large surface area in the limited space available for chemical sensing, due to the expansive maxilloturbinal. Morphologically, the white-tailed deer is shown to possess a dorsal meatus that leads to an olfactory recess, a nasal architecture that has been shown in other non-primate species to cause unique nasal airflow patterns to develop during sniffing that are optimized for odorant delivery to the sensory part of the nose. Additionally, we demonstrate that, during respiration, airflow in the nasal vestibule and the anterior maxilloturbinal region may be transitional or turbulent, in which case turbulent mixing is expected to enhance respiratory heat and moisture exchange, which could be an important contribution to thermoregulation and water conservation in the white-tailed deer.

Decepción,surprise,colèreetfuria

This article presents a corpus-based approach to the comparison of partial synonyms on an intra- and cross-linguistic level. The methodology is demonstrated using the example of three lexical fields in French and Spanish, all from the domain of the lexis of emotion: surprise, disappointment, anger. After the intralinguistic analysis of French surprise/étonnement/stupeur and Spanish decepción/desencanto/desilusión/desengaño, the investigation is extended to include a cross-linguistic comparison of French colère/fureur with Spanish cólera/furia. As a first step, data that reveals the combinatory behaviour of the analysed lexical items is extracted from a corpus containing comparable quantities of French and Spanish literary and newspaper texts. The co-occurrences thus obtained are then divided into categories of lexical dimensions, e. g. intensity (furia desatada, honda decepción) or causation of emotion (déchaîner la colère, provoquer de la surprise), creating a «tertium comparationis» that allows for the comparison between partial synonyms or presumed equivalents in different languages. Finally, the actual analysis is performed using EmoGrammes – an instrument based on descriptive statistics allowing to aggregate and represent the results graphically. This heuristic method has proven particularly helpful in the complex folds of cross-linguistic comparison.

Folding pathway of a multidomain protein depends on its topology of domain connectivity

How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant.

Nobel prizes and nature's surprises

A Magician of Virology from Australia A Divided Nobel Prize and a New Era in Immunology More Nobel Prizes in Immunology Immunity, Infections and Transplantations Transgressing Borders in Science and Scenes of Life Making Sense of Hearing: Art and Science Unraveling the Complexity of Protein Folding It's So Beautiful, You See, So Beautiful Coda.

RNA structural analysis by evolving SHAPE chemistry.

RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base-pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2'-hydroxyl group in the ribose ring, and the reactivity of the 2'-hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2'-hydroxyl is reactive at single-stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base-pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2' hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2'- hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome-wide exploration of RNA structure and function.

Misfolding of Luciferase at the Single‐Molecule Level

AbstractThe folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding.

Misfolding of luciferase at the single-molecule level.

The folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding.

The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long-lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Refolding of mechanically denatured HIV-1-PR monomers is also a multiple-pathway process. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, and provide information regarding possible structural features of the PF state.

Building a Surface Atlas of Hippocampal Subfields from MRI Scans using FreeSurfer, FIRST and SPHARM.

The hippocampus is widely studied with neuroimaging techniques given its importance in learning and memory and its potential as a biomarker for brain disorders such as Alzheimer's disease and epilepsy. However, its complex folding anatomy often presents analytical challenges. In particular, the critical hippocampal subfield information is usually ignored by hippocampal registration in detailed morphometric studies. Such an approach is thus inadequate to accurately characterize hippocampal morphometry and effectively identify hippocampal structural changes related to different conditions. To bridge this gap, we present our initial effort towards building a computational framework for subfield-guided hippocampal morphometry. This initial effort is focused on surface-based morphometry and aims to build a surface atlas of hippocampal subfields. Using the FreeSurfer software package, we obtain valuable hippocampal subfield information. Using the FIRST software package, we extract reliable hippocampal surface information. Using SPHARM, we develop an approach to create an atlas by mapping interpolated subfield information onto an average surface. The empirical result using ADNI data demonstrates the promise and good reproducibility of the proposed method.

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts.

A rotamer library to enable modeling and design of peptoid foldamers.

Peptoids are a family of synthetic oligomers composed of N-substituted glycine units. Along with other “foldamer” systems, peptoid oligomer sequences can be predictably designed to form a variety of stable secondary structures. It is not yet evident if foldamer design can be extended to reliably create tertiary structure features that mimic more complex biomolecular folds and functions. Computational modeling and prediction of peptoid conformations will likely play a critical role in enabling complex biomimetic designs. We introduce a computational approach to provide accurate conformational and energetic parameters for peptoid side chains needed for successful modeling and design. We find that peptoids can be described by a “rotamer” treatment, similar to that established for proteins, in which the peptoid side chains display rotational isomerism to populate discrete regions of the conformational landscape. Because of the insufficient number of solved peptoid structures, we have calculated the relative energies of side-chain conformational states to provide a backbone-dependent (BBD) rotamer library for a set of 54 different peptoid side chains. We evaluated two rotamer library development methods that employ quantum mechanics (QM) and/or molecular mechanics (MM) energy calculations to identify side-chain rotamers. We show by comparison to experimental peptoid structures that both methods provide an accurate prediction of peptoid side chain placements in folded peptoid oligomers and at protein interfaces. We have incorporated our peptoid rotamer libraries into ROSETTA, a molecular design package previously validated in the context of protein design and structure prediction.

Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases.

Structural and Mechanistic Insights into the PAPS-Independent Sulfotransfer Catalyzed by Bacterial Aryl Sulfotransferase and the Role of the DsbL/DsbI System in Its Folding

Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.

Hydrophobic supplements in cell‐free systems: Designing artificial environments for membrane proteins

Membrane proteins (MPs) are of central interest for the pharmaceutical industry but their production is usually a challenging task. The complex folding mechanisms of newly synthesized MPs often require interactions with specific compounds for improved stability. Conditions for the production of high‐quality samples are therefore difficult to predict and frequently cannot be provided in conventional protein expression platforms. Cell‐free (CF) biosynthetic systems allow, in contrast to living cells, nonrestricted access to the protein production machinery. Reaction conditions can be adjusted according to particular requirements and modified by supplementing single additives or even cocktails of compounds. These options have initiated completely new research fields for the cotranslational stabilization and folding of MPs in artificial environments. Based on established and efficient CF production protocols, a recent focus was to explore and define suitable supplements for CF expression reactions that are useful for the generation of high‐quality MP samples. Besides classical detergents and lipids, a variety of new compounds with interesting properties have been discovered and synthesized. We compile the currently available toolbox for MP solubilization in CF systems and summarize new developments and perspectives for the directed modulation of CF biosynthetic environments.

Substitution of dietary fish oil with plant oils is associated with shortened mid intestinal folds in Atlantic salmon (Salmo salar)

BackgroundFish meal and fish oil are increasingly replaced by ingredients from terrestrial sources in the feeds for farmed salmonids due to expanding production and reduced availability of marine feed raw material. Fish oil that is rich in n-3 polyunsaturated fatty acids is considered beneficial to human health in general and to prevent intestinal inflammation and carcinogenesis in particular. In contrast, n-6 fatty acids that are present in many vegetable oils have been associated with increased risk of colitis and colon cancer in rodents and humans, as well as lowered transcription levels of certain stress and antioxidant-related genes in Atlantic salmon.The aim of the present study was to investigate the intestinal health in Atlantic salmon fed with different vegetable oils as partial substitutes of fish oil in the diet. A feed trial lasting for 28 weeks included one reference diet containing fish oil as the sole lipid source and three diets where 80% of the fish oil was replaced by a plant oil blend with either olive oil, rapeseed oil or soybean oil as the main lipid source. These plant oils have intermediate or low n-3/n-6-ratios compared to fish oil having a high n-3/n-6-ratio. The protein and carbohydrate fractions were identical in all the feeds.ResultsMorphometric measurements showed significantly shorter folds in the mid intestine in all groups fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological and immunohistochemical examination did not show clear difference in the degree of inflammation or proliferation of epithelial cells related to dietary groups, which was further confirmed by real-time RT-PCR which revealed only moderate alterations in the mRNA transcript levels of selected immune-related genes.ConclusionsShortened intestinal folds might be associated with reduced intestinal surface and impaired nutrient absorption and growth, but our results suggest that partial substitution of dietary fish oil with vegetable oils does not have any major negative impact on the intestinal health of Atlantic salmon.

ATP Acts as Switch for Toggling Calreticulin Between its Lectin and Chaperone Function

Calreticulin (CRT) is a lectin-binding chaperone that plays an important role in the assembly and folding of the major histocompatibility complex (MHC) class I proteins that present antigenic peptides on the cell surface and enable their recognition by T-cells. CRT binds to both specific oligosaccharide structures on MHC class I during their folding as well as a polypeptide part of MHC and thus plays a crucial role in stabilizing folding intermediates, preventing aggregation, and allowing the MHC protein to attain its native structure. Recent experiments have shown that the initial interaction between CRT and a monoglucosylated MHC protein is glycan-driven with glycan-independent interactions representing a second step in the chaperone cycle of CRT. Still, what factors trigger a switch from glycan-dependent to glycan-independent mode of interactions between CRT and its substrate is not well understood. Through computational investigations involving molecular dynamics simulations and structure based molecular docking, we show that ATP-binding serves as a switch for toggling CRT between two distinct modes of interactions. Specifically, the binding of ATP on CRT at the location that is distant from the glycan binding site leads to reduction in the affinity of CRT toward glycans and thus acts as a switch that induces a disengagement of a glycan from CRT, and induces exposure of hydrophobic regions on the surface of the globular domain allowing CRT to act as a chaperone through favoring interactions with the polypeptide component of the MHC. Further, employing community network analysis we have predicted residues that participate in allosteric signaling between ATP and glycan binding site of CRT. Modifying these residues has a large effect on the communication pathways in CRT consistent with experiments and support our model of ATP as a switch for CRT function.

Contribution of protein phosphorylation to binding-induced folding of the SLBP-histone mRNA complex probed by phosphorus-31 NMR

Contribution of protein phosphorylation to binding-induced folding of the SLBP-histone mRNA complex probed by phosphorus-31 NMR

Actinous enigma or enigmatic actin: Folding, structure, and functions of the most abundant eukaryotic protein.

Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins.

Unraveling the Mystery of Ribosome Induced RNA Unfolding

During translation, the ribosome encounters various structures in the mRNA from simple hairpins to more complex tertiary RNA folds. These structures must be unwound for accurate decoding and translation to proceed. To probe the unwinding activity of the ribosome, we prepared dye labeled RNA transcripts and dye labeled ribosomes to directly report on the folded/unfolded states of the RNA at various stages along the translational elongation cycle. Consistent with previous studies, we found that the ribosome was sufficient to resolve RNA structures in the absence of any additional factors. Surprisingly, significant unwinding was observed in the absence of translocation or motion of the ribosome along the mRNA and can be attributed to dynamic motions within and between the two ribosomal subunits. This suggests that thermal energy resident within these ribosomal motions is sufficient to lower the energetic barrier of unfolding. The extent of unwinding was observed to depend on the relative stability of the RNA structure used and the number of tRNAs present in the ribosome. Differences between unfolding of these RNA structures in the presence and absence of the ribosome will be discussed.