In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound ...nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp
interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63-94 residues. Longer RNCs (apoHmp
) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.
Kinetic trapping in protein folding Varela, Angela E; England, Kevin A; Cavagnero, Silvia
Protein engineering, design and selection,
12/2019, Letnik:
32, Številka:
2
Journal Article
Recenzirano
The founding principles of protein folding introduced by Christian Anfinsen, together with the numerous mechanistic investigations that followed, assume that protein folding is a thermodynamically ...controlled process. On the other hand, this review underscores the fact that thermodynamic control is far from being the norm in protein folding, as long as one considers an extended chemical-potential landscape encompassing aggregates, in addition to native, unfolded and intermediate states. Here, we highlight the key role of kinetic trapping of the protein native state relative to unfolded, intermediate and, most importantly, aggregated states. We propose that kinetic trapping serves an important role in biology by protecting the bioactive states of a large number of proteins from deleterious aggregation. In the event that undesired aggregates were somehow formed, specialized intracellular disaggregation machines have evolved to convert any aberrant populations back to the native state, thus restoring a fully bioactive and aggregation-protected protein cohort.
Anfinsen’s thermodynamic hypothesis does not explicitly take into account the possibility of protein aggregation. Here, we introduce a cyclic-perturbation approach to prove that not only the native ...state but also soluble aggregates of most proteins can be highly populated under mild, physiologically relevant conditions, even at very low concentration. Surprisingly, these aggregates are not necessarily amyloid in nature and are usually not observed in bioactive proteins due to the extremely low kinetic flux from the native state toward a region of the chemical-potential landscape encoding aggregates. We first illustrate this concept for the representative model protein apomyoglobinat room temperature and no denaturantand demonstrate kinetic trapping of the native state relative to at least two different types of soluble, predominantly nonamyloid aggregates. The concentration and temperature dependence of aggregation confirm the above scenario. Extension of our analysis to the Escherichia coli proteome shows that the majority of the soluble bacterial proteome is also kinetically trapped in the nonaggregated state. Hence, the existence and low kinetic accessibility of large aggregates at room temperature and pH 6–7 is a general phenomenon. We also show that the average critical protein concentration for aggregation of most of the bacterial proteome is extremely small, much lower than the typical cellular protein concentration. Hence, the thermodynamic driving force for protein aggregation is large even if aggregation does not usually occur in healthy cells due to kinetic trapping. A broader view of Anfinsen’s thermodynamic hypothesis encompassing all protein states, including aggregates, is necessary to understand the behavior of proteins in their natural environment.
We still know very little about protein folding in the cell and what parameters and molecular machines cause soluble proteins to achieve their native conformation. Although Anfinsen’s thermodynamic ...hypothesis explains the origin of protein stability, it does not account for the presence of protein aggregates and where they lie in an energy landscape relative to the native state, under physiologically relevant conditions. Protein folding starts within the ribosome during biosynthesis. While the ribosome and cotranslationally active molecular chaperones help nascent chains achieve their native state, little is known about the influence of nascent polypeptides and proteins on the ribosome and its apparent stability. In this thesis I show that Anfinsen’s thermodynamic hypothesis needs to be modified to include soluble and insoluble protein aggregates because most proteins are kinetically trapped relative to their aggregated states. In addition, I explore some of the determinants of the apparent stability of the 70S ribosome in E. coli.This thesis is divided into four chapters. Chapter 1 explains Anfinsen’s thermodynamic hypothesis together with its major implications, and outlines the major aspects of the ribosome structure and function. This chapter serves as the conceptual background for the remaining portions of this document.Chapters 2 and 3 explore the concept of protein kinetic trapping under physiologically relevant conditions and demonstrates the need to extend Anfinsen’s hypothesis to include protein aggregates. More specifically, Chapter 2 introduces an experimental strategy denoted as the “cyclic perturbation approach” to show that two structurally distinct, non-amyloid aggregate states of sperm whale apomyoglobin are kinetically trapped relative to each other and relative to the native state. The experiments presented in Chapter 3 show that kinetic trapping is a more general phenomenon than previously thought, and that it can be extended to the large majority of soluble proteins in E. coli. A series of kinetic simulations confirms this phenomenon.Chapter 4 showcases the influence of the nascent chain on the apparent stability of the 70S E. coli ribosome. The presence of the newly synthesized polypeptide was found to stabilize the ribosome. Surprisingly, characteristic properties of the nascent chain, including its N- and C-terminal residues, net charge, and length (beyond 32 residues) do not have any influence on the structural stabilization of the 70S complex by the nascent polypeptide. This chapter also introduces a working model for denaturant-induced ribosomal disassembly.Overall, this dissertation shows that the kinetic trapping of native, unfolded and intermediate protein states relative to aggregates under physiologically relevant conditions is a general phenomenon in Nature. In addition, it shows that nascent proteins contribute to increasing the apparent stability of the bacterial ribosome.
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Bacteriophytochromes (Bphs) are bacterial red‐light photoreceptors found in photosynthetic and non‐photosynthetic bacteria. Bphs undergo reversible photoconversion between two distinct ...red (Pr) and far‐red (Pfr) light absorbing states. An organic cofactor, biliverdin (BV) is required for Pr/Pfr photoconversion. Bphs are composed of a photosensory core (PC) including PAS, GAF and PHY domains and an effector domain, usually a histidine kinase. We report on the preliminary crystal structure of the unusual Bph from Rhodopseudomonas palustris, RpBphP3 (P3) PC bound to BV in the Pr state. Unlike classical Bphs, P3 photoconverts between Pr and near‐red (Pnr) states. P3 crystals (30–60 □m in all dimensions) yielded diffraction at 100 K to a maximum resolution of 2.8 Å. P3 crystals are in P212121 space group with unit cell dimensions of a=155.92 Å, b=185.32 Å, and c=199.53 Å. The initial P3 structure was determined with the molecular replacement (MR) method implemented in HKL3000 using the crystal structure of P3 lacking PHY domain (PDB ID code
2OOL
) as search model. P3 crystallized as a dimer, which is physiologically relevant oligomeric state of the protein. Informed by structural and sequence analyses, we identify amino acids in the BV‐binding pocket of P3 and at the dimer interface that play a role in the unusual photochemistry and fluorescence of this protein.
The essential guide to successful couples therapy at every stage of the lifecycleA variety of therapeutic interventions can help couples develop the tools for a successful relationship. Yet many ...practitioners begin seeing couples without extensive training in couples work. To fill this gap in their therapeutic repertoires, noted couples therapist Michele Harway brings together other well-known experts in marriage and family therapy to offer the Handbook of Couples Therapy, a comprehensive guide to the study and practice of couples therapy.The book's chapters provide a variety of perspectives along developmental, theoretical, and situational lines. Recognizing the need for clinically proven, evidence-based approaches, chapters provide detailed coverage of the most effective treatment modes. Couples at different stages of the lifecycle feature prominently in the text, as do relevant special issues and treatment approaches for each stage.Subjects covered include: Premarital counseling from the PAIRS perspective (an extensive curriculum of interventions for premarital couples) The first years of marital commitment Couples with young children Couples with adolescents Therapy with older couples Same sex couples A variety of theoretical approaches, including Cognitive-Behavioral, Object Relational, Narrative, Integrative, and Feminist and Contextual Special issues and situations, including serious illness, physical aggression, addiction, infidelity, and religious/spiritual commitments or conflicts Providing a diverse set of treatment approaches suited to working with a wide range of adult populations, the Handbook of Couples Therapyis an essential resource for mental health professionals working with couples.
Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC ...cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5− tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.
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•rRNA and proteins in CRCs are produced in dedicated tumor domains•Differentiated tumor cells experience an irreversible loss of biosynthetic capacities•POLR1A-high CRC cells reside at the top of the tumor cell hierarchy•Both LGR5+ and LGR5− tumor cells within biosynthetic niches fuel tumor growth
Morral and colleagues discovered that most rRNA and proteins synthesized in colorectal cancers (CRCs) are contributed by a limited subset of tumor cells that reside adjacent to the stroma. This architecture defines a common stem cell hierarchy. In some CRCs, the biosynthetic tumor cell population renders LGR5+ tumor cells dispensable.
Body-mass index (BMI) has increased steadily in most countries in parallel with a rise in the proportion of the population who live in cities
. This has led to a widely reported view that ...urbanization is one of the most important drivers of the global rise in obesity
. Here we use 2,009 population-based studies, with measurements of height and weight in more than 112 million adults, to report national, regional and global trends in mean BMI segregated by place of residence (a rural or urban area) from 1985 to 2017. We show that, contrary to the dominant paradigm, more than 55% of the global rise in mean BMI from 1985 to 2017-and more than 80% in some low- and middle-income regions-was due to increases in BMI in rural areas. This large contribution stems from the fact that, with the exception of women in sub-Saharan Africa, BMI is increasing at the same rate or faster in rural areas than in cities in low- and middle-income regions. These trends have in turn resulted in a closing-and in some countries reversal-of the gap in BMI between urban and rural areas in low- and middle-income countries, especially for women. In high-income and industrialized countries, we noted a persistently higher rural BMI, especially for women. There is an urgent need for an integrated approach to rural nutrition that enhances financial and physical access to healthy foods, to avoid replacing the rural undernutrition disadvantage in poor countries with a more general malnutrition disadvantage that entails excessive consumption of low-quality calories.
Comparable global data on health and nutrition of school-aged children and adolescents are scarce. We aimed to estimate age trajectories and time trends in mean height and mean body-mass index (BMI), ...which measures weight gain beyond what is expected from height gain, for school-aged children and adolescents.
For this pooled analysis, we used a database of cardiometabolic risk factors collated by the Non-Communicable Disease Risk Factor Collaboration. We applied a Bayesian hierarchical model to estimate trends from 1985 to 2019 in mean height and mean BMI in 1-year age groups for ages 5–19 years. The model allowed for non-linear changes over time in mean height and mean BMI and for non-linear changes with age of children and adolescents, including periods of rapid growth during adolescence.
We pooled data from 2181 population-based studies, with measurements of height and weight in 65 million participants in 200 countries and territories. In 2019, we estimated a difference of 20 cm or higher in mean height of 19-year-old adolescents between countries with the tallest populations (the Netherlands, Montenegro, Estonia, and Bosnia and Herzegovina for boys; and the Netherlands, Montenegro, Denmark, and Iceland for girls) and those with the shortest populations (Timor-Leste, Laos, Solomon Islands, and Papua New Guinea for boys; and Guatemala, Bangladesh, Nepal, and Timor-Leste for girls). In the same year, the difference between the highest mean BMI (in Pacific island countries, Kuwait, Bahrain, The Bahamas, Chile, the USA, and New Zealand for both boys and girls and in South Africa for girls) and lowest mean BMI (in India, Bangladesh, Timor-Leste, Ethiopia, and Chad for boys and girls; and in Japan and Romania for girls) was approximately 9–10 kg/m2. In some countries, children aged 5 years started with healthier height or BMI than the global median and, in some cases, as healthy as the best performing countries, but they became progressively less healthy compared with their comparators as they grew older by not growing as tall (eg, boys in Austria and Barbados, and girls in Belgium and Puerto Rico) or gaining too much weight for their height (eg, girls and boys in Kuwait, Bahrain, Fiji, Jamaica, and Mexico; and girls in South Africa and New Zealand). In other countries, growing children overtook the height of their comparators (eg, Latvia, Czech Republic, Morocco, and Iran) or curbed their weight gain (eg, Italy, France, and Croatia) in late childhood and adolescence. When changes in both height and BMI were considered, girls in South Korea, Vietnam, Saudi Arabia, Turkey, and some central Asian countries (eg, Armenia and Azerbaijan), and boys in central and western Europe (eg, Portugal, Denmark, Poland, and Montenegro) had the healthiest changes in anthropometric status over the past 3·5 decades because, compared with children and adolescents in other countries, they had a much larger gain in height than they did in BMI. The unhealthiest changes—gaining too little height, too much weight for their height compared with children in other countries, or both—occurred in many countries in sub-Saharan Africa, New Zealand, and the USA for boys and girls; in Malaysia and some Pacific island nations for boys; and in Mexico for girls.
The height and BMI trajectories over age and time of school-aged children and adolescents are highly variable across countries, which indicates heterogeneous nutritional quality and lifelong health advantages and risks.
Wellcome Trust, AstraZeneca Young Health Programme, EU.
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