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The functional mechanisms of multidomain proteins often exploit interdomain interactions, or “cross-talk.” An example is human Pin1, an essential mitotic regulator consisting of a Trp–Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-μs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW–PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

Feminist solutions to the climate crisis 8 March 2022 — 11:00AM TO 12:00PM Anonymous (not verified) 1 March 2022 Online

This International Women’s Day panel explores intersectional feminist approaches to solving the climate crisis.

Gender inequality and climate change are closely intertwined. Due to their different and unequal social roles and status, women, girls and people of marginalised genders are disproportionately affected by climate change impacts, also differentiated by factors such as age, race, ability and location.

They are also leading innovative solutions to climate change at all levels, especially in frontline communities. Yet as the Glasgow Women’s Leadership statement highlighted at COP26, there is still a lack of momentum for prioritising their knowledge, tools and leadership in climate policy and action.  

This panel considers what feminist approaches can bring to climate change communication and movement building, spotlighting a range of local and regional gender-responsive climate initiatives. It also discusses concrete examples of feminist and gender-responsive policies to address climate change, such as the Feminist Green New Deal.

The panel highlights the critically important work being carried out by diverse feminist activists and researchers, and how the audience can centre gender and social equality in their work on climate change.  

Duterte’s Victory Is Cry for Help From Those Left Behind in Philippines Expert comment sysadmin 12 May 2016

But large support for mainstream parties and a mature democratic system should keep the country from slipping back towards authoritarianism.

The victory of political outsider Rodrigo Duterte in the 2016 Philippines’ elections is proof that a significant minority of the country’s population feels left behind by its recent economic success and estranged from its political elite. However the results of the elections as a whole suggest that most voters opted for a continuation of the current government’s policies.

Duterte looks almost certain to be inaugurated as the next president of the Philippines on 30 June. The country’s presidential voting system – a single round, first-past-the-post election – delivered victory to a populist outsider with 39 per cent support. Two candidates advocating a continuation of the current government’s policies − the Liberal Party’s Mar Roxas and independent Grace Poe − polled a combined 45 per cent. The long-standing factionalism within Philippines elite politics split the ‘anti-Duterte’ vote.

Changing the conversation

The contrast between Duterte and Roxas could hardly be greater. Mar Roxas is the grandson of the first president of an independent Philippines, a graduate of Wharton Business School and a former investment banker in the US. Rodrigo Duterte is a political outsider with an electoral base geographically almost as far from Manila as is possible to get in the Philippines: the city of Davao on the island of Mindanao.

The story of Duterte’s victory is the story of how ‘Duterte managed to change the national conversation from poverty towards crime and corruption,’ says Marites Vitug, editor-at-large of one of the Philippines’ most popular online news sites, Rappler. In January, Duterte was running fourth in opinion polls but a strategy that positioned him as the only opponent to the Manila elite gave him victory. This is the first time a provincial official has made it to the top job.

The headline figures tell us that the Philippines’ economy has done very well under President Benigno Aquino. Between 2010 and 2014, growth averaged 6.3 per cent per year. That fell to a still-impressive 5.8 per cent last year but is expected to pick up this year and next, according to the Asian Development Bank. Growth in agriculture, however, is significantly slower and rural areas feel left behind. While economic growth is benefiting the majority, inequality is worsening and resentment rising in poor villages. The contrast between the metropolitan sophistication of the Makati district in Manila and life in faraway provinces such as Duterte’s Mindanao is widening.

Ironically the Philippines’ economic success is a part of the explanation for the defeat of the ‘mainstream’ presidential candidates. Crime and corruption may have become more important issues simply because more voters have become better off and therefore more likely to be concerned about crime and corruption than before. It’s also undeniable that Duterte has a record for getting things done. Human rights groups rightly criticize his (at best) tolerance of the extra-judicial killing of alleged criminals but his repeated re-election as mayor demonstrates that many citizens are prepared to accept that in exchange for improved personal security. A surprising number of Manila residents have actually moved to Davao because of its better quality of life.

Traditional power bases

However, the results as a whole suggest a narrow majority in favour of current policies. In the vice-presidential race, the Liberal Party candidate Leni Robredo is narrowly ahead of Ferdinand ‘Bongbong’ Marcos, the son of the eponymous former president. Like Duterte she is regarded as a successful mayor of a well-run city, Albay. Duterte’s running mate Alan Cayetano received just 14 per cent of the vote.

In the senate election, Liberals won five of the 12 seats being contested, with a party- backed independent winning a sixth. The opposition, even with boxing champion and national idol Manny Pacquiao running for the United Nationalist Alliance, won four.

Taken as a whole, the results show the enduring nature of traditional Philippines power bases. The country’s many islands and distinct linguistic and cultural regions are virtual fiefs in which families and big bosses can wield almost total power through control of local authorities, businesses, the courts and security forces.

Threat to democracy?

It’s easy to forget that the election of Ferdinand Marcos in 1965 was originally welcomed as a challenge to the traditional elites of Philippine politics. The same accolades are currently greeting Duterte. Could they presage a return to the Philippines’ bad old days?

This seems less likely. Philippine democracy has matured considerably since Marcos declared martial law in 1972. There is a substantial, and vocal, middle class with experience of mobilizing against ‘bad’ presidents. There will also be pressures from international investors and the Philippines’ treaty ally, the United States, for better governance.

The Philippines will chair the Association of Southeast Asian Nations next year. That will put Duterte in the international spotlight as host of several international meetings – including the East Asia Summit attended by, among others, the presidents of China, Russia and the US. Since his victory Duterte has promised to act with decorum in office and declared that his election campaign antics were just a ploy to attract attention. Some leaders in Southeast Asia will use his victory to buttress their arguments against allowing their people to freely vote. It’s up to Duterte to decide whether he wants to be an advertisement for – or an argument against – democracy.

To comment on this article, please contact Chatham House Feedback

Root Causes of Rohingya Crisis Must Not be Ignored Expert comment sysadmin 28 September 2017

The focus on Aung San Suu Kyi masks the complete lack of an adequate response to the crisis in Myanmar, whether at the global or regional level.

Aung San Suu Kyi’s recent speech on the Rohingya crisis was - at best - light on details on how the current situation could be remedied and - at worst - full of easily disproven assertions.

While she does not directly control the military, it is her government that is blocking humanitarian access to the areas affected by the violence. And the Rohingya have faced systemic persecution and discrimination for decades.

Some may argue this is simply realpolitik and that any public support for the Rohingya could mean facing a backlash from the military and a large part of her support base. But arguably, she does have moral authority (which helped her in the past to stand up to the military generals) that is now being eroded by her ambivalence in speaking out.

However, the focus on Aung San Suu Kyi masks the complete lack of an adequate response to the crisis – whether at the global or regional level.

Undertaking dangerous and perilous journeys

Since the attacks on border and military posts by the armed group Arakan Rohingya Salvation Army (ARSA) in August 2017, there has been a strong military crackdown against the Rohingya in Rakhine state: a substantial number of Rohingya villages destroyed; close to half a million people Rohingya fleeing into Bangladesh and tens of thousands internally displaced within Myanmar.

Prince bin Ra’ad , UN High Commissioner for Human Rights, has called the crisis a “textbook example of ethnic cleansing” and tens of thousands of Rohingya are still undertaking dangerous and perilous journeys seeking sanctuary. In response, the UN and EU have focused on addressing the immediate humanitarian crisis in Bangladesh, which is already home to many formerly displaced Rohingya communities.

Within the region, Sheikh Hasina’s secular Awami League government in Bangladesh initially proposed joint military operations with Myanmar against the ARSA - in part because of concerns about the long standing relationship between Rohingya political or armed groups and the Jamaat-e-Islami, an ally of the main opposition Bangladesh Nationalist Party (BNP).

However, faced with massive refugee flows, Bangladesh turned its focus to the humanitarian crisis while stressing that Myanmar must allow the return of refugees. Bangladesh’s concern is partly motivated by internal security concerns. If the current situation becomes protracted, with no clear resolution in sight, frustration could create the conditions for further radicalisation within Rohingya communities.

The Association of South East Asian Nations (ASEAN) has yet to come to grips with the situation. Despite its mandate to ensure peace and stability within the region, its policy of non-interference and consensus trumps the need to secure and maintain stability. Instead countries have responded bilaterally - for example, Indonesia sent its foreign minister to both Myanmar and Bangladesh while Malaysia has been consistently vocal about its concerns.

So this raises broader questions on the effectiveness of ASEAN. Currently celebrating its 50th anniversary, ASEAN needs to decide how to mediate and resolve issues with regional implications as its principle of non-intervention effectively blocks any constructive discussion on the Rohingyas ongoing statelessness and impact of this on the region.

However, there is also an opportunity here for ASEAN to consider how mediation and negotiation could potentially manage such crises. And there is a historical precedent: the 1989 Comprehensive Plan of Action on Indo-Chinese refugees saw cooperation between recipient countries in the region and the international community on how to resettle Vietnamese refugees (although Cold War considerations did play a part in that specific crisis).

India and China have both backed Myanmar, reflecting their economic and security interests in the country but also motivated by each wanting to contain the influence of the other within Myanmar. Rakhine is important with its natural resources and coastal location and, as China is not directly affected by the refugee crisis, it has less to lose than others in standing by Aung San Suu Kyi and her government.

India is nearing completion of the Kaladan Multi-modal Transit Transport Project, connecting the Bay of Bengal with the northeast Indian state of Mizoram, and sees Myanmar as an important market for its regional ambitions. For parts of the Indian administration, the crisis plays to a domestic narrative that some of the Rohingya already settled in Jammu and Kashmir have links to armed groups in Pakistan and are an internal security concern.

So while India is providing humanitarian aid to Bangladesh, it is also threatening to deport almost 40,000 Rohingya. The case is currently being heard at the Indian Supreme Court, but given that the Rohingya lack citizenship in Myanmar, it is not clear to where they would be deported.

Myanmar, ASEAN and other affected countries need to show political will to find a solution to the Rohingya’s long-standing issue of statelessness - discrimination was legally formalised in a 1982 Burma Citizenship Law, which recognised 135 ethnicities for citizenship but excluded the Rohingya.

The root causes of this crisis – long standing discrimination, persecution and lack of citizenship – cannot be ignored. There is a need for a comprehensive peace process, which recognises the ethnic and religious diversity within Myanmar.

And incentives, such as improving infrastructure, access to services and livelihoods, may also be needed to ensure there is a lasting solution that allows the Rohingya return and thrive as part of Myanmar society.

Without such a response, it is difficult to see an end to the current impasse.

Putting the Hong Kong Crisis into Historical and Comparative Perspective 14 November 2019 — 8:30AM TO 9:30AM Anonymous (not verified) 17 October 2019 Chatham House | 10 St James's Square | London | SW1Y 4LE

This roundtable will focus on current events unfolding in Hong Kong, where the territory has been convulsed with protests for several months.

The speakers will examine how class, race and poverty play into the conflict. Taking a comparative approach, they will examine the generational divide, looking at the ideological gulf between the older, more conservative and pro-Beijing population versus the younger, more pro-democracy protesters. The discussion will also draw upon the erosion of trust between police and the wider public.

While acknowledging the unique features of this wave of unrest, the speakers will draw parallels, placing the current crisis in Hong Kong beside events that have occurred in other periods and other places.

Parallels to be explored include those with Shanghai struggles of the 1910s through 1980s and upheavals and crackdowns in the former Soviet bloc during the Cold War.

The Rohingya Crisis: Three Years On 17 September 2020 — 1:30PM TO 2:15PM Anonymous (not verified) 9 September 2020 Online

Speakers examine the current situation of the Rohingya people and assess the threat that COVID-19 poses to the health and human rights of refugees and displaced people.

It has been three years since a military-led crackdown forced more than 740,000 Rohingya to flee across the border into Bangladesh to escape collective punishment and violence in Myanmar.

Most refugees have sought shelter in Bangladesh’s Cox’s Bazar district, where access to clean water is limited, sanitation facilities are lacking, and due to overcrowding, social distancing is impossible.

While the number of reported COVID-19 cases has so far been relatively low, testing capacity remains limited and anecdotal reports from humanitarians suggest that COVID-19 has spread extensively through the refugee camps and the Bangladeshi host community.

The speakers also consider the different approaches taken by neighbouring states, regional and international organizations in responding to the crisis.

What can be done to address the needs of refugees in the short term and how can fundamental human rights be restored and protected during the time of COVID-19? What aid provision has been successfully delivered within Rakhine State and in what ways?

Ahead of elections in Myanmar in November, how can the international community persuade the Myanmarese government into positive action? And what would a sustainable solution to the Rohingya crisis look like and what are the competing views over how such a solution should be delivered?

This event is held in partnership with The Atlantic Council.

Torin Greenwood, Jonathan Kariv and Noah Williams
Math. Comp. 93 (), 2959-2983.
Abstract, references and article information

Constantin Carle and Marlis Hochbruck
Math. Comp. 93 (), 2611-2641.
Abstract, references and article information

Algorithms can be very useful, but lately, with so much data being created and shared, and with the increase in their use in critical areas such as hiring, credit, and health care, algorithms are under intense scrutiny about their fairness. People experience the effects of an algorithm's conclusion, but the data and steps that form the basis for that conclusion are frequently hidden from them (as if inside a black box). Cathy O'Neil talks about the unfairness of most predictive algorithms.

Math is often described as the science of patterns, which makes it a natural subject to help in the study of the underlying causes of patterns found in nature, for example, bands of vegetation that often occur on gently sloped terrains in certain near-desert ecosystems worldwide. We are starting to learn more about these bands' common properties by using mathematical models built on data, such as rainfall totals and the curvature of the terrain. Mary Silber talks about these mathematical models of vegetation bands.

Christian Weiss
Theor. Probability and Math. Statist. 111 (), 199-209.
Abstract, references and article information

Marko Voutilainen, Lauri Viitasaari and Pauliina Ilmonen
Theor. Probability and Math. Statist. 111 (), 181-197.
Abstract, references and article information

Olha Prykhodko and Kostiantyn Ralchenko
Theor. Probability and Math. Statist. 111 (), 123-135.
Abstract, references and article information

Lasse L. Wolf and Hong-Wei Zhang
Proc. Amer. Math. Soc. 152 (), 5445-5453.
Abstract, references and article information

Xiaoshang Jin
Proc. Amer. Math. Soc. 152 (), 5327-5337.
Abstract, references and article information

Yong Suk Moon
Proc. Amer. Math. Soc. 152 (), 5095-5103.
Abstract, references and article information

Donghyeok Lim and Christian Maire
Proc. Amer. Math. Soc. 152 (), 5013-5024.
Abstract, references and article information

Naoki Endo and Shiro Goto
Proc. Amer. Math. Soc. 152 (), 5007-5011.
Abstract, references and article information

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Recent studies have suggested that innate immune responses exhibit characteristics associated with memory linked to modulations in both vertebrates and invertebrates. However, the diverse evolutionary paths taken, particularly within the invertebrate taxa, should lead to similarly diverse innate immunity memory processes. Our understanding of innate immune memory in invertebrates primarily comes from studies of the fruit fly Drosophila melanogaster, the generality of which is unclear. Caenorhabditis elegans typically inhabits soil harboring a variety of fatal microbial pathogens; for this invertebrate, the innate immune system and aversive behavior are the major defensive strategies against microbial infection. However, their characteristics of immunological memory remains infantile. Here we discovered an immunological memory that promoted avoidance and suppressed innate immunity during reinfection with bacteria, which we revealed to be specific to the previously exposed pathogens. During this trade-off switch of avoidance and innate immunity, the chemosensory neurons AWB and ADF modulated production of serotonin and dopamine, which in turn decreased expression of the innate immunity-associated genes and led to enhanced avoidance via the downstream insulin-like pathway. Therefore, our current study profiles the immune memories during C. elegans reinfected by pathogenic bacteria and further reveals that the chemosensory neurons, the neurotransmitter(s), and their associated molecular signaling pathways are responsible for a trade-off switch between the two immunological memories.

Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre–mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC–MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics, and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM's CTD can discriminate against Mn2+, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn2+ incorporation.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance. One such strategy involves the TipA class of transcription factors, which constitute minimal autoregulated multidrug resistance (MDR) systems against diverse antibiotics. However, we have insufficient information regarding how antibiotic binding induces transcriptional activation to design molecules that could interfere with this process. To learn more, we determined the crystal structure of SkgA from Caulobacter crescentus as a representative TipA protein. We identified an unexpected spatial orientation and location of the antibiotic-binding TipAS effector domain in the apo state. We observed that the α6–α7 region of the TipAS domain, which is canonically responsible for forming the lid of antibiotic-binding cleft to tightly enclose the bound antibiotic, is involved in the dimeric interface and stabilized via interaction with the DNA-binding domain in the apo state. Further structural and biochemical analyses demonstrated that the unliganded TipAS domain sterically hinders promoter DNA binding but undergoes a remarkable conformational shift upon antibiotic binding to release this autoinhibition via a switch of its α6–α7 region. Hence, the promoters for MDR genes including tipA and RNA polymerases become available for transcription, enabling efficient antibiotic resistance. These insights into the molecular mechanism of activation of TipA proteins advance our understanding of TipA proteins, as well as bacterial MDR systems, and may provide important clues to block bacterial resistance.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

The life cycle of malaria parasites in both their mammalian host and mosquito vector consists of multiple developmental stages that ensure proper replication and progeny survival. The transition between these stages is fueled by nutrients scavenged from the host and fed into specialized metabolic pathways of the parasite. One such pathway is used by Plasmodium falciparum, which causes the most severe form of human malaria, to synthesize its major phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Much is known about the enzymes involved in the synthesis of these phospholipids, and recent advances in genetic engineering, single-cell RNA-Seq analyses, and drug screening have provided new perspectives on the importance of some of these enzymes in parasite development and sexual differentiation and have identified targets for the development of new antimalarial drugs. This Minireview focuses on two phospholipid biosynthesis enzymes of P. falciparum that catalyze phosphoethanolamine transmethylation (PfPMT) and phosphatidylserine decarboxylation (PfPSD) during the blood stages of the parasite. We also discuss our current understanding of the biochemical, structural, and biological functions of these enzymes and highlight efforts to use them as antimalarial drug targets.

13 July 2020

26 August 2020

Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like de novo lipogenesis to glucose availability in many cell types is carbohydrate response element–binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes. Here we report that ChREBP is modified by proline hydroxylation at several residues. Proline hydroxylation targets both ectopically expressed ChREBP in cells and endogenous ChREBP in mouse liver. Functionally, we found that specific hydroxylated prolines were dispensable for protein stability but required for the adequate activation of ChREBP upon exposure to high glucose. Accordingly, ChREBP target gene expression was rescued by re-expressing WT but not ChREBP that lacks hydroxylated prolines in ChREBP-deleted hepatocytes. Thus, proline hydroxylation of ChREBP is a novel post-translational modification that may allow for therapeutic interference in metabolic diseases.

The development of a chronic, low-grade inflammation originating from adipose tissue in obese subjects is widely recognized to induce insulin resistance, leading to the development of type 2 diabetes. The adipose tissue microenvironment drives specific metabolic reprogramming of adipose tissue macrophages, contributing to the induction of tissue inflammation. Uncoupling protein 2 (UCP2), a mitochondrial anion carrier, is thought to separately modulate inflammatory and metabolic processes in macrophages and is up-regulated in macrophages in the context of obesity and diabetes. Here, we investigate the role of UCP2 in macrophage activation in the context of obesity-induced adipose tissue inflammation and insulin resistance. Using a myeloid-specific knockout of UCP2 (Ucp2ΔLysM), we found that UCP2 deficiency significantly increases glycolysis and oxidative respiration, both unstimulated and after inflammatory conditions. Strikingly, fatty acid loading abolished the metabolic differences between Ucp2ΔLysM macrophages and their floxed controls. Furthermore, Ucp2ΔLysM macrophages show attenuated pro-inflammatory responses toward Toll-like receptor-2 and -4 stimulation. To test the relevance of macrophage-specific Ucp2 deletion in vivo, Ucp2ΔLysM and Ucp2fl/fl mice were rendered obese and insulin resistant through high-fat feeding. Although no differences in adipose tissue inflammation or insulin resistance was found between the two genotypes, adipose tissue macrophages isolated from diet-induced obese Ucp2ΔLysM mice showed decreased TNFα secretion after ex vivo lipopolysaccharide stimulation compared with their Ucp2fl/fl littermates. Together, these results demonstrate that although UCP2 regulates both metabolism and the inflammatory response of macrophages, its activity is not crucial in shaping macrophage activation in the adipose tissue during obesity-induced insulin resistance.