Invitation Only Research Event

Event participants

Denise Brown, United Nations Deputy Special Representative of the Secretary-General, Resident and Humanitarian Coordinator in the Central African Republic
Chair: Ben Shepherd, Consulting Fellow, Africa Programme, Chatham House

Research Event

Event participants

Mohamed Guleid, Chief Executive Officer, Frontier Counties Development Council
Nuradin Dirie, Chair, Puntland Presidential Advisory Council
Aden Abdi, Horn of Africa Programme Director, Conciliation Resources
Chair: Dr Zahbia Yousuf, Senior Research Advisor, Saferworld 

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.

The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

Collaborating with the public is the key for a more engaging government experience.

2 March 2020

Henry J. Pownall
May 1, 2020; 61:595-597
Commentary

This week our host Scott Carey catches up with Sean Bradley and Dominic Preston fresh off the back of the International Consumer Electronics Show (CES) in Las Vegas.

Dom explains why he was surprised, both in a good and bad way, by the latest foldable screen technology and Sean talks about what is going on in the gaming laptop space, as Alienware looks to soften its image.

Then Dom talks about a piece of connected wood and the gang break down the big controversy from the show floor regarding a certain innovative dildo.

We are also announcing the sad news that the UK Tech Weekly Podcast will be going on an indefinite hiatus while we reassess our podcasting output. We would like to thank anyone that has taken the time to listen to us for these one hundred and twelve (112!) episodes and rest assured we will be back in some guise soon.

See acast.com/privacy for privacy and opt-out information.

Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity due to the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

1 October 2008 , Number 11

Nuclear, biological or chemical weapons and acts of terror may make the headlines, but it is conventional arms that take the lives in large numbers; maybe around a thousand a day. This month, a United Nations committee will try to find a way to limit the arms trade with a new treaty. For those facing the barrel of a gun, it cannot come a moment too soon.

4 December 2019

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

Ceramides (Cers) with ultralong (~32-carbon) chains and -esterified linoleic acid, composing a subclass called omega-O-acylceramides (acylCers), are indispensable components of the skin barrier. Normal barriers typically contain acylCer concentrations of ~10 mol%; diminished concentrations, along with altered or missing long periodicity lamellar phase (LPP), and increased permeability accompany an array of skin disorders, including atopic dermatitis, psoriasis, and ichthyoses. We developed model membranes to investigate the effects of the acylCer structure and concentration on skin lipid organization and permeability. The model membrane systems contained six to nine Cer subclasses as well as fatty acids, cholesterol, and cholesterol sulfate; acylCer content—namely, acylCers containing sphingosine (Cer EOS), dihydrosphingosine (Cer EOdS), and phytosphingosine (Cer EOP) ranged from zero to 30 mol%. Systems with normal physiologic concentrations of acylCer mixture mimicked the permeability and nanostructure of human skin lipids (with regard to LPP, chain order, and lateral packing). The models also showed that the sphingoid base in acylCer significantly affects the membrane architecture and permeability and that Cer EOP, notably, is a weaker barrier component than Cer EOS and Cer EOdS. Membranes with diminished or missing acylCers displayed some of the hallmarks of diseased skin lipid barriers (i.e., lack of LPP, less ordered lipids, less orthorhombic chain packing, and increased permeability). These results could inform the rational design of new and improved strategies for the barrier-targeted treatment of skin diseases.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

Lipid rafts are organized plasma membrane microdomains, which provide a distinct level of regulation of cellular metabolism and response to extracellular stimuli, affecting a diverse range of physiologic and pathologic processes. This Thematic Review Series focuses on Biology of Lipid Rafts rather than on their composition or structure. The aim is to provide an overview of ideas on how lipid rafts are involved in regulation of different pathways and how they interact with other layers of metabolic regulation. Articles in the series will review the involvement of lipid rafts in regulation of hematopoiesis, production of extracellular vesicles, host interaction with infection, and the development and progression of cancer, neuroinflammation, and neurodegeneration, as well as the current outlook on therapeutic targeting of lipid rafts.

Over the past couple seasons, the Rays have preached patience as the organization provided time for the top prospects to make it up to the Majors. Now, the focus has been primarily in remaining flexible and keeping positions open for the young talent arriving from the Minors.

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

To ensure fetal lipid supply, maternal blood triglyceride (TG) concentrations are robustly elevated during pregnancy. Interestingly, a lower increase in maternal blood TG concentrations has been observed in some obese mothers. We have shown that high-fat (HF) feeding during pregnancy significantly reduces maternal blood TG levels. Therefore, we performed this study to investigate if and how obesity alters maternal blood TG levels. Maternal obesity was established by prepregnant HF feeding (ppHF), which avoided the dietary effect during pregnancy. We found that maternal blood TG concentrations in ppHF dams were not only remarkably lower than control dams, but the TG peak occurred earlier during gestation. Hepatic TG production and intestinal TG absorption were unchanged in ppHF dams, but systemic lipoprotein lipase (LPL) activity was increased, suggesting that increased blood TG clearance contributes to the decreased blood TG concentrations in ppHF dams. Although significantly higher levels of UCP1 protein were observed in iBAT of ppHF dams, Ucp1 gene deletion did not restore blood TG concentrations in ppHF dams. Expression of the angiopoietin-like protein 4 (ANGPTL4), a potent endogenous LPL inhibitor, was significantly increased during pregnancy. However, the pregnancy-induced elevation of blood TG was almost abolished in Angptl4^-/- dams. Compared with control dams, Angptl4 mRNA levels were significantly lower in iBAT, gWAT and livers of ppHF dams. Importantly, ectopic overexpression of ANGPTL4 restored maternal blood TG concentrations in ppHF dams. Together, these results indicate that ANGPTL4 plays a vital role in increasing maternal blood TG concentrations during pregnancy. Obesity impairs the rise of maternal blood TG concentrations by reducing ANGPTL4 expression in mice.

α-Klotho is a circulating factor with well-documented anti-aging properties; however, the central role of α-klotho in metabolism remains largely unexplored. The current study investigated the potential role of central α-klotho to modulate NPY/AgRP neurons, energy balance, and glucose homeostasis. Intracerebroventricular (ICV) administration of α-klotho suppressed food intake, improved glucose profiles, and reduced body weight in mouse models of Type I and II diabetes. Furthermore, central α-klotho inhibition via an anti-α-klotho antibody impaired glucose tolerance. Ex vivo patch clamp electrophysiology and immunohistochemical analysis revealed that α-klotho suppresses NPY/AgRP neuron activity, at least in part, by enhancing mIPSC’s. Experiments in hypothalamic GT1-7 cells observed α-klotho induces phosphorylation of AKT^ser473, ERK^{thr202/tyr204}, and FOXO1^ser256, as well as blunts AgRP gene transcription. Mechanistically, fibroblast growth factor 1 (FGFR1) inhibition abolished the downstream signaling of α-klotho, negated its ability to modulate NPY/AgRP neurons, and blunted its therapeutic effects. PI3 kinase inhibition also abolished α-klotho’s ability to suppress food intake and improve glucose clearance. These results indicate a prominent role of hypothalamic α-klotho/FGFR1/PI3K signaling in the modulation of NPY/AgRP neuron activity and maintenance of energy homeostasis, thus providing new insight into the pathophysiology of metabolic disease.

The brain mechanisms underlying the association of hyperglycemia with depressive symptoms are unknown. We hypothesized that disrupted glutamate metabolism in pregenual anterior cingulate cortex (ACC) in type 1 diabetes (T1D) without depression affects emotional processing. Using proton magnetic resonance spectroscopy (MRS), we measured glutamate concentrations in ACC and occipital cortex (OCC) in 13 T1D without major depression (HbA1c=7.1±0.7% [54±7mmol/mol]) and 11 healthy non-diabetic controls (HbA1c=5.5±0.2% [37±3mmol/mol]) during fasting euglycemia (EU) followed by a 60-minute +5.5mmol/l hyperglycemic clamp (HG). Intrinsic neuronal activity was assessed using resting-state blood oxygen level dependent functional MRI to measure the fractional amplitude of low frequency fluctuations in slow-band 4 (fALFF4). Emotional processing and depressive symptoms were assessed using emotional tasks (Emotional-Stroop, Self-Referent-Encoding-Task SRET) and clinical ratings (HAM-D, SCL-90-R), respectively. During HG, ACC glutamate increased (1.2mmol/kg, +10%, p=0.014) while ACC fALFF4 was unchanged (-0.007, -2%, p=0.449) in T1D; in contrast, glutamate was unchanged (-0.2mmol/kg, -2%, p=0.578) while fALFF4 decreased (-0.05, -13%, p=0.002) in controls. OCC glutamate and fALFF4 were unchanged in both groups. T1D had longer SRET negative-word response-times (p=0.017) and higher depression-rating scores (HAM-D p=0.020; SCL-90-R-depression p=0.008). Higher glutamate change tended to associate with longer Emotional-Stroop response-times in T1D only. Brain glutamate must be tightly controlled during hyperglycemia due to the risk for neurotoxicity with excessive levels. Results suggest that ACC glutamate control mechanisms are disrupted in T1D, which affects glutamatergic neurotransmission related to emotional or cognitive processing. Increased prefrontal glutamate during acute hyperglycemic episodes could explain our previous findings of associations between chronic hyperglycemia, cortical thinning and depressive symptoms in T1D.

Hyperglycemia is a potent regulator of endogenous glucose production (EGP). Loss of this ‘glucose effectiveness’ is a major contributor to elevated plasma glucose concentrations in type 2 diabetes (T2D). ATP-sensitive potassium channels (K_ATP channels) in the central nervous system (CNS) have been shown to regulate EGP in humans and rodents. We examined the contribution of central K_ATP channels to glucose effectiveness. Under fixed hormonal conditions (‘pancreatic clamp’ studies), hyperglycemia suppressed EGP by ~50% in both non-diabetic humans and normal Sprague Dawley rats. By contrast, antagonism of K_ATP channels with glyburide significantly reduced the EGP-lowering effect of hyperglycemia in both humans and rats. Furthermore, the effects of glyburide on EGP and gluconeogenic enzymes in rats were abolished by intracerebroventricular (ICV) administration of the KATP channel agonist diazoxide. These findings indicate that about half of EGP suppression by hyperglycemia is mediated by central K_ATP channels. These central mechanisms may offer a novel therapeutic target for improving glycemic control in T2D.

A greater decrease in 24-h energy expenditure (24EE) during 24h fasting defines a thriftier metabolic phenotype prone to weight gain during overfeeding and resistant to weight loss during caloric restriction. As the thermogenic response to mild cold exposure (COLD) may similarly characterize this human phenotype identified by acute fasting conditions, we analyzed changes in 24EE and sleeping metabolic rate (SLEEP) in a whole-room indirect calorimeter during 24h fasting at thermoneutrality (24°C) and during energy balance both at thermoneutrality (24°C) and mild cold (19°C) in 20 healthy volunteers (80% male, age: 36.6±11.4y, percentage body fat: 34.8±10.5%). Greater decrease in 24EE during fasting (thriftier phenotype) was associated with less increase in 24EE during COLD, i.e. less cold-induced thermogenesis. Greater decreases in plasma fibroblast growth factor 21 (FGF21) after 24h fasting and after COLD were highly correlated and associated with greater decreases in SLEEP in both conditions. We conclude that the metabolic responses to short-term fasting and COLD are associated and mediated by the liver-derived hormone FGF21. Thus, the 24EE response to COLD further identifies the thrifty versus spendthrift phenotype, providing an additional setting to investigate the physiological mechanisms underlying the human metabolic phenotype and characterizing the individual susceptibility to weight change.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed “templated folding,” whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Barbara H. Braffett
May 1, 2020; 69:1000-1010
Complications

Mandeep Bajaj
Nov 1, 2005; 54:3148-3153
Metabolism

Mariam Alatrach
Apr 1, 2020; 69:681-688
Pathophysiology

Patrice D. Cani
Jun 1, 2008; 57:1470-1481
Metabolism

Mary C. Gannon
Sep 1, 2004; 53:2375-2382
Pathophysiology

1 October 2008 , Number 11

Nuclear, biological or chemical weapons and acts of terror may make the headlines, but it is conventional arms that take the lives in large numbers; maybe around a thousand a day. This month, a United Nations committee will try to find a way to limit the arms trade with a new treaty. For those facing the barrel of a gun, it cannot come a moment too soon.

Invitation Only Research Event

Event participants

Anita Isaacs, Professor of Political Science, Haverford College; Co-Director, Migration Encounters Project
Juan Ricardo Ortega, Principal Advisor for Central America, Inter-American Development Bank
Chair: Amy Pope, Associate Fellow, Chatham House; US Deputy Homeland Security Adviser for the Obama Administration (2015-17)

4 December 2019