Understanding the frustration arising from the competition between function,misfolding, and aggregation in a globular protein

Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron–sulfur clusters but whose mutations are associated with Friedreich’s ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.Frustration is a general condition that arises in the presence of conflicting requirements. A system is frustrated when it is impossible to fully minimize its energy by optimizing simultaneously all of the possible interactions among its components (1). Although complex systems tend in general to exhibit frustration because of the large number and heterogeneity of their components, protein molecules are remarkable in that their folding process involves interactions that express a minimal level of frustration. According to the so-called principle of minimal frustration, the energy of proteins decreases as they explore conformations increasingly similar in structure to the native state (2). Consequently, the free energy landscape of proteins is characterized by the presence of a well-defined global minimum and very few other local minima, which are typically intermediate states along the folding pathway. This organization of conformational space normally ensures rapid and reliable folding (2–6).Proteins, however, have evolved not only to fold, but also to function. Because the evolutionary constraints that select for a given function may be in conflict with the folding process, it is possible that local frustration patterns may localize in specific regions of proteins, in particular in their active sites. Indeed, a statistical survey of different proteins has shown that frustrated interactions tend to cluster at binding sites and that such frustration decreases upon complex formation (7). Because frustration is associated with the presence of local minima in the free energy landscape, it is important to understand how proteins have evolved to minimize the possible effects associated with these local minima, which are likely to contain misfolded elements and thus to potentially give rise to aggregation.To address this question we studied frataxin, a mitochondrial protein that binds both Fe²⁺ and Fe³⁺ ions and forms a ternary complex with the two main components of the iron–sulfur cluster biogenesis machinery (8–11). This protein offers good opportunities for investigating the relationships between folding, misfolding, and disease. Indeed, its dysfunction is related to a neurodegenerative disease called Friedreich’s ataxia (12). Frataxin is also capable of binding different divalent and trivalent cations, whose recognition sites have been mapped (13). Furthermore, frataxin is involved in donating iron to ferrochelatease via direct interaction through an extended binding site involving some of the residues implicated in metal binding (14).We have previously shown that frataxin folds via a complex mechanism, which we described through a broad free energy barrier (15). This feature, which has been associated with frustration (16), allows the experimental characterization of both the early and late events of folding (16–19). In this work we explored the mechanistic details of the folding reaction of frataxin at residue-level resolution. This result was achieved by characterizing the structures of both the early and late events of folding using Φ-value analysis (20) and restrained molecular dynamics simulations (21). By analyzing the structures of the different states along the folding process we found an unexpected number of nonnative interactions that slow down folding and superpose with the highly frustrated regions, as detected by the frustratometer server (22). The nonnative regions, which display peculiar Φ values, either negative or greater than unity, were predicted on the basis of the transition state structures determined from the Φ values, and subsequently confirmed by a second round of amino acid substitutions rationally designed to probe misfolded regions along the folding pathway.The characterization of the folding pathway of frataxin and of its misfolded elements enables us to discuss the competition between folding and function and its consequences for misfolding and aggregation.     

Morphological assessment of pancreatic islet hormone content following aerobic exercise training in rats with poorly controlled Type 1 diabetes mellitus

Regular exercise has been shown to improve many complications of Type 1 diabetes mellitus (T1DM) including enhanced glucose tolerance and increased cardiac function. While exercise training has been shown to increase insulin content in pancreatic islets of rats with T1DM, experimental models were severely hyperglycemic and not undergoing insulin treatment. Further, research to date has yet to determine how exercise training alters glucagon content in pancreatic islets. The purpose of the present investigation was to determine the impact of a 10-week aerobic training program on pancreatic islet composition in insulin-treated rats with T1DM. Second, it was determined whether the acute, exercise-mediated reduction in blood glucose experienced in rats with T1DM would become larger in magnitude following aerobic exercise training. Diabetes was induced in male Sprague-Dawley rats by multiple low dose injections of streptozotocin (20mg/kg i.p.) and moderate intensity aerobic exercise training was performed on a motorized treadmill for one hour per day for a total of 10 weeks. Rats with T1DM demonstrated significantly less islet insulin, and significantly more islet glucagon hormone content compared with non-T1DM rats, which did not significantly change following aerobic training. The reduction in blood glucose in response to a single exercise bout was similar across 10 weeks of training. Results also support the view that different subpopulations of islets exist, as small islets (<50 μm diameter) had significantly more insulin and glucagon in rats with and without T1DM.     

Treatment of refractory adult lymphoblastic leukemia (ALL) with 4'(9- acridinylamino) methanesulfon-M-anisidide (AMSA)

Twenty-four adults with ALL were treated with AMSA alone or in combination. Twenty-two were treated at time of relapse and two patients after failing primary induction therapy. All had been treated with anthracyclines prior to receiving AMSA. Of the 22 patients with ALL in relapse, 4 achieved a complete remission. Two of these patients have relapsed while receiving maintenance chemotherapy; one died 1 mo after achieving remission due to the occurrence of cholycystitis in the setting of pancytopenia and one patient underwent bone marrow transplantation and is in remission at 8 mo after the second remission. Both patients who failed primary induction therapy remain in remission at 11 and 36 mo, respectively. The use of AMSA should be considered for patients with ALL who fail primary induction as well as those whose leukemia becomes resistant to conventional agents.     

Detection of circulating crosslinked fibrin derivatives by a heat extraction-SDS gradient gel electrophoretic technique

A technique has been developed to identify and quantitate unique plasmic degradation products of crosslinked fibrin in plasma. In this method, fibrin derivatives are extracted by heat precipitation and dissolved with disulfide bond reduction, after which the crosslinked gamma-gamma chain remnants are identified by SDS-polyacrylamide gradient gel electrophoresis and quantitated by densitometric analysis. A heterogenous group of gamma-gamma chains with molecular weights between 100,000 and 76,000 daltons was identified in lysates of crosslinked fibrin during plasmic degradation in vitro. Three stages of crosslinked fibrin degradation have been arbitrarily defined based primarily on the extent of degradation of these gamma-gamma polypeptide chains. As little as 20 microgram of crosslinked fibrin digests added to 1 ml of normal plasma could be detected by the heat-extraction--gel- electrophoresis technique, identifying the gamma-gamma derivatives with molecular weights of 96,000, 86,000, 82,000, and 76,000 daltons. Plasmic derivatives of gamma-gamma chains were not found in normal plasma, but they were identified in the plasma of patients with disseminated intravascular coagulation and deep-vein thrombosis, both before and in increased quantity during successful thrombolytic therapy.     

Chemoattractant-induced changes in surface expression and redistribution of a functional ligand for P-selectin on neutrophils

Adhesion between platelets and neutrophils is mediated through the interaction of P-selectin on activated platelets with a carbohydrate- containing structure on neutrophils, and occurs under both static and shear conditions. Recent studies using flow chambers have shown that neutrophils become activated after binding to surface-adherent platelets expressing P-selectin. The objective of the present study was to investigate the effect of such activation on the interactions of platelet P-selectin with its ligand on neutrophils. Flow cytometric analyses using P-selectin chimeras revealed that activation induced a rapid and marked reduction in chimera binding, with levels of binding decreased by 71% after 15 minutes of stimulation with the chemotactic agent, FMLP. Using a visual assay of platelet-neutrophil rosetting, we showed that the P-selectin ligand was translocated and clustered at the uropod of neutrophils following the shape changes and polarization induced by chemotactic stimulation. Activated neutrophils bound to surface-adherent platelets also displayed the clustering of P-selectin ligand at the uropod, and these neutrophils detached from the platelets when a shear stress (2 dynes/cm2) was applied through the adhesion chamber. These results indicate that chemotactic stimulation of neutrophils induces changes in the surface expression and distribution of a biologically relevant ligand for P-selectin, and that these changes might influence the adhesive interactions occurring between neutrophils and activated platelets.     

Acute lymphoblastic leukemia of childhood: identification of two distinct regions of deletion on the short arm of chromosome 12 in the region of TEL and KIP1

Cytogenetic analysis of acute lymphoblastic leukemia (ALL) of childhood identified nonrandom chromosomal abnormalities of the short arm of chromosome 12. The alterations include deletions that are thought to be indicative of the presence of a tumor suppressor gene that is mutated on the remaining allele. To refine further the chromosomal localization of this gene, we analyzed the loss of heterozygosity (LOH) of chromosome 12 in 100 primary ALL samples using 22 polymorphic markers and identified two distinct smallest common deleted regions on chromosome 12p13. One region is flanked by D12S77 and D12S98 and has a size of 4 cM. Twenty-six percent of informative patients showed LOH in this region. This region may contain the TEL gene. The other region is flanked by D12S269 and D12S308 including the KIP1 gene. Forty-four percent of informative patients showed LOH in this second region. Mutational analysis of KIP1 using polymerase chain reaction-single- strand conformation polymorphism analysis and Southern blot analysis showed no homozygous deletions and point mutations suggesting that the altered gene in this second region is not the KIP1. Clinical data showed that LOH of 12p was demonstrated more frequently in precursor-B ALLs (32 of 80; 40%) than in T-ALLs (1 of 20; 5%) (P = .0027). Furthermore, patients with 12p LOH were younger (P = .013), with a lower DNA index (P = .046), but they had the same survival rates at 3 years. In summary, these data suggest that two different tumor suppressor genes are on chromosome arm 12p, which act separately in the development of childhood precursor-B ALLs. One of the tumor suppressor genes is in the region the KIP1 gene, but our data suggest this gene is not abnormal. The other target is in the region of the TEL gene; and this candidate deserves further study.     

Clinical features and outcome in childhood T-cell leukemia-lymphoma according to stage of thymocyte differentiation: a Pediatric Oncology Group Study

The immunophenotypes of lymphoblasts from children with newly diagnosed T-cell acute lymphoid leukemia (T-ALL, n = 101) or T-cell non-Hodgkin lymphoma (T-NHL, n = 31) were analyzed to correlate stage of thymocyte differentiation with clinical features and outcome. The 67 boys and 34 girls with T-ALL were 1 month to 18 years old (median, 8 years) with leukocyte counts ranging from 2 to 810 x 10(9)/L (median, 55 x 10(9)/L). Eighteen of these patients were black, and 70 had a mediastinal mass. Twenty-six boys and five girls with a median age of 9 years (range, 1 to 20 years) had T-NHL. Seven of these patients were black, and 24 had a mediastinal mass. The distributions of thymocyte developmental stages (early [CD7+], intermediate [CD1+ and/or CD4+ and/or CD8+], and mature [CD3+]) in cases of T-ALL and T-NHL were significantly different: 34%, 43%, and 23% v 6%, 62%, and 32% (P = .02). A comparison of the patients' clinical features according to the maturational stage of thymocytes failed to disclose significant differences in the majority of characteristics studied. However, patients with mature-stage T-NHL, with or without the addition of subjects with mature-stage T-ALL, were less likely to have a mediastinal mass (P = .02 for both comparisons). Those with intermediate-stage T-cell malignancy (T-ALL and T-NHL combined) were the subgroup most likely to have a mediastinal mass (P = .01). Response to remission induction therapy was significantly worse in the T-ALL subgroup with an early-stage phenotype: a failure rate of 21% v 0% and 6% for the two more differentiated phenotypic subgroups (P = .007). Event-free survival was not affected by thymocyte maturational stage in cases of either T-ALL or T-NHL. Despite evidence of clinical heterogeneity among the maturational stages of T-cell malignancies in children, these developmental subdivisions do not appear to be critical determinants of outcome once remission is achieved. We conclude that such phenotypes need not be included in the stratification plans for clinical trials using common induction treatment.     

Secrets,shame and guilt: HIV disclosure in rural Chinese families from the perspective of caregivers

This in-depth ethnographic study examines the processes, barriers and impacts experienced by Mainland Chinese caregivers residing in rural Yunnan for disclosing HIV status to their adolescents born with the infection and other community members. In particular, highlighted are their associated personal, social, relational and cultural vulnerabilities. A purposeful sample of 13 pairs of HIV-born adolescents between the ages of 11–19 years and their primary caregivers were recruited in December 2014. The Conceptual Model Sexual Health Disclosure (CMSHD) was modified with Chinese culturally specific adaptation to guide the exploration of the complex aspects of the adolescents and caregiver’s relationships. All interviews were audiotaped, transcribed verbatim and then translated into English.

Complex sociocultural interactions emerging in these dyadic interviews included guilt, shame, unintended disclosure, the need for secrecy, perceived stigma, ‘loss of face’, protection of parents, inability to support their families and deep concerns over lineage and future marriage prospects amongst the parents, which had significant psychosocial and even physical impacts for these adolescents born with HIV and their families. Frontline health-care workers and HIV peer support programs are recommended to counteract China’s current top-down biomedical disease-related approach to health services. It is crucial to provide instrumental avenues of confidential psychosocial support including disclosure approaches for caregivers of HIV positive adolescents within a uniquely Chinese cultural context.     

Aven‐mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in conventional osteosarcoma

Conventional high‐grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis‐free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti‐apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G₂ cell‐cycle arrest; Chk1 protein levels were attenuated and ATR–Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven‐depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven‐controlled ATR–Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix‐embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR–Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Four cancer cases after esophageal atresia repair: Time to start screening the upper gastrointestinal tract

Esophageal atresia(EA) is one of the most common congenital digestive malformations and requires surgical correction early in life. Dedicated centers have reported survival rates up to 95%. The most frequent comorbidities after EA repair are dysphagia(72%) and gastroesophageal reflux(GER)(67%). Chronic GER after EA repair might lead to mucosal damage, esophageal stricturing, Barrett's esophagus and eventually esophageal adenocarcinoma. Several long-term follow-up studies found an increased risk of Barrett's esophagus and esophageal carcinoma in EA patients, both at a relatively young age. Given these findings, the recent ESPGHAN-NASPGHAN guideline recommends routine endoscopy in adults born with EA. We report a series of four EA patients who developed a carcinoma of the gastrointestinal tract: three esophageal carcinoma and one colorectal carcinoma in a colonic interposition. These cases emphasize the importance of lifelong screening of the upper gastrointestinal tract in EA patients.