Member’s question time: Is Russia losing the South Caucasus? 22 October 2024 — 1:00PM TO 1:30PM Anonymous (not verified) 2 September 2024 Online

Join us and ask Chatham House Senior Research Fellow, Natalie Sabanadze anything about the situation in the Caucasus. Submit your questions in advance.

Whilst Russia focuses on its illegal invasion of Ukraine, the situation at its southern border is evolving. Relations between the three states in the South Caucasus and Moscow have never been easy as Russia tried to maintain its dominance by leveraging vulnerabilities, playing one side against another to keep conflicts simmering and even engaging in open military aggression.  Although the violence seen in the 1990s and early 2000s has abated, the war in Ukraine has had an indirect impact on the region, bringing a change to the status quo.

Russia abandoned its long-standing support for Armenia, allowing for the collapse of Nagorny-Karabakh and the restoration of the territorial integrity of Azerbaijan with the backing of Turkey. As a result, Azerbaijan has emerged as a dominant regional player with Baku recently declaring its interest to join BRICS.  Turkey’s influence has grown, while Armenia frustrated by Russia’s change of heart has been turning cautiously towards the EU and the US.

In Georgia, meanwhile, the ruling party has been consolidating its grasp on power, rolling back democratic reforms and pivoting away from the West. Georgia’s long-awaited European integration process has been suspended, following the adoption of the Russian-style foreign agents legislation.

Join us as our Senior Research Fellow in the Russia and Eurasia Programme answers your questions in this quick-fire session assessing the extent to which the Russian influence has changed since the start of the invasion of Ukraine and who is there to fill the vacuum; how geopolitical contestation in the region is going to impact aspirations of Armenia, Azerbaijan and Georgia; and much more.

Submit your questions to Natalie Sabanadze in advance of the event. Your questions will drive the conversation.

In conversation with Speaker Emerita Nancy Pelosi 14 October 2024 — 12:00PM TO 1:00PM Anonymous (not verified) 19 September 2024 Chatham House and Online

The former Speaker of the House of Representatives discusses challenges to American democracy and the importance of leadership in an election year.

As the US gears up for the 2024 Presidential election, the importance of leadership in politics has come to the fore. As the Harris and Trump campaigns move towards election day, the heightened sense of strong leadership is central to the position of both candidates.

Following her first election to the House in 1987, Speaker Emerita Nancy Pelosi has been central to American political leadership for over two decades. Serving as the 52nd Speaker of the House of Representative, Speaker Pelosi rose to become one of the most powerful and consequential Speakers in American political history.

Following the release of her recent book The Art of Power, Speaker Pelosi will discuss how she became a master legislator, working with numerous presidents, her political legacy in the house and her leadership during a contentious time in US politics.

Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin–tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T's N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin–tropomyosin binding and weaker tropomyosin–actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin–thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM.

Ligand bias is the ability of ligands to differentially activate certain receptor signaling responses compared with others. It reflects differences in the responses of a receptor to specific ligands and has implications for the development of highly specific therapeutics. Whereas ligand bias has been studied primarily for G protein–coupled receptors (GPCRs), there are also reports of ligand bias for receptor tyrosine kinases (RTKs). However, the understanding of RTK ligand bias is lagging behind the knowledge of GPCR ligand bias. In this review, we highlight how protocols that were developed to study GPCR signaling can be used to identify and quantify RTK ligand bias. We also introduce an operational model that can provide insights into the biophysical basis of RTK activation and ligand bias. Finally, we discuss possible mechanisms underpinning RTK ligand bias. Thus, this review serves as a primer for researchers interested in investigating ligand bias in RTK signaling.

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations in IP3Rs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IP3R function, and how the perturbation of as-sociated calcium signals contribute to the pathogenesis and severity of these diseases remains largely uncharacterized. Moreover, many of these diseases occur as the result of autosomal dominant inheritance, suggesting that WT and mutant subunits associate in heterotetrameric channels. How the in-corporation of different numbers of mutant subunits within the tetrameric channels affects its activities and results in different disease phenotypes is also unclear. In this report, we investigated representative disease-associated missense mutations to determine their effects on IP3R channel activity. Additionally, we designed concatenated IP3R constructs to create tetrameric channels with a predefined subunit composition to explore the functionality of heteromeric channels. Using calcium imaging techniques to assess IP3R channel function, we observed that all the mutations studied resulted in severely attenuated Ca2+ release when expressed as homotetramers. However, some heterotetramers retained varied degrees of function dependent on the composition of the tetramer. Our findings suggest that the effect of mutations depends on the location of the mutation in the IP3R structure, as well as on the stoichiometry of mutant subunits assembled within the tetrameric channel. These studies provide insight into the pathogenesis and penetrance of these devastating human diseases.

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

Nancy Pelosi in Taiwan: What lies ahead for China and the US? Expert comment GBhardwaj 3 August 2022

Chatham House experts examine the implications for Taiwan, China and the United States of Nancy Pelosi’s visit to the island.

China’s fading ties with Washington?

Dr Yu Jie

US House Speaker, Nancy Pelosi’s, visit to Taiwan has plunged China-US relations into a new low as the reservoir of trust forged between the two sides over the last 40 years appears to be almost exhausted.

However, her move will likely not result in the full-scale crisis across the Taiwan strait that some hawkish voices in both Beijing and Washington believe. Instead, Beijing will most likely offer a combination of military posturing toward the US navy and economic sanctions on Taiwanese agricultural and manufacturing products in order to send a clear bellwether to any future potential visits by high-level Western political figures.

Neither Beijing nor Washington has declared a willingness to change the current status quo as the present impasse benefits both governments – but for different reasons. For China, the best approach is to reach a military and economic capability that prevents US engagement with Taiwan without the use of force. For the US, the strategic ambiguity under the Taiwan Relations Act remains an effective card to counter China’s growing military influence in the Indo-Pacific and keep itself relevant within the region as a security guarantor. Yet, both sides have decided to kick the issue of Taiwan’s status down the road, believing that time is ultimately on their side.

Despite a chorus of nationalistic rhetoric, China will be careful not to stumble into an accidental conflict which risks colossal damage on all fronts. Chinese President, Xi Jinping, must weigh all of the options before him as Beijing cannot afford to be perceived as unilaterally seeking to change what it agreed with the US back in 1979 when ties were re-established. If that happens, it will provoke the US political establishment to reach a unanimous agreement to change its ‘One China Policy’ and, ahead of the 20th Communist Party Congress where Xi is expected to be crowned for a historic third term, the last thing he wants is an unnecessary conflict with Taiwan.

The road to escalation?

Dr Bill Hayton

Beijing has chosen to take issue over Nancy Pelosi’s visit to Taiwan in a way that it did not do for other recent US Congressional visits to the island. Several high-ranking US senators visited in April and May this year yet none of these visits triggered the prospect of a cross-strait crisis. So why has Beijing chosen to turn Pelosi’s visit into a stand-off?

Pelosi’s visit is part of a performance in which both actors – the US and China – are playing primarily for their domestic audiences. This comes at a time when ruling circles in Beijing are preparing for the five-yearly Communist Party Congress. Amid a slowing economy and successive COVID-19 variants, Xi Jinping cannot afford to look weak as he prepares the ground for his third term of office. Meanwhile, the US, represented either by President Joe Biden or House Speaker Pelosi, cannot back down at this point without looking weak itself.

Both sides have moved military assets into strategic positions near Taiwan to demonstrate their resolve. Neither side wants confrontation yet neither wishes to be humiliated. Currently, Pelosi’s visit, amid posturing by China, will make the US appear strong, but the consequences are likely to play out over a longer period. Xi Jinping will need to appear to have recaptured the initiative between now and the congress in the autumn when the risk of an incident will be at its greatest.

Taiwan controls several isolated islands that could be pressured by Chinese forces in the event of a future crisis. The Kinmen and Matsu archipelagos lie just a few miles off the coast of the mainland and have been at the centre of previous confrontations. There are also two other points of concern. Pratas Island – known as Dongsha – sits halfway between Taiwan and Hong Kong. Itu Aba – known as Taiping – is the largest of the Spratly Islands in the centre of the South China Sea. All would be vulnerable to an attack by the People’s Liberation Army, the principal military force of China, and the armed wing of the Chinese Communist Party.

A military confrontation between China and the US over Taiwan, or further south in the South China Sea, would have major impacts on regional and global trade. An estimated $300 billion worth of trade passes through the area every month. Japan and South Korea depend heavily on flows of oil and gas through the sea. Exports from Vietnam, Malaysia, Indonesia and the Philippines would also be heavily affected by disruptions to shipping, increased insurance costs and interruptions in inflows of raw materials. The impact on a world economy already suffering major disruption because of the Russian invasion of Ukraine and the lingering effects of COVID-19 would indeed be stark.

Is a shift in US policy on the cards?

Dr Leslie Vinjamuri

Nancy Pelosi’s visit to Taiwan will undoubtedly be seen as a provocation by Beijing – even if it should not be. Pelosi’s trip to the Indo-Pacific, which also includes visits to Singapore, Malaysia, South Korea and Japan, comes at a time of growing tension between the US and China in the region.

It also comes at a time when the divide among Washington’s foreign policy elite is growing, with some arguing that it is time to abandon the country’s policy of ‘strategic ambiguity’, where it refrains from stating how it would react were China to openly and deliberately attack Taiwan. Indeed, recent statements by President Joe Biden have raised questions about whether the US is set to make a policy change. But, since both its ‘One China Policy’ and policy of strategic ambiguity have been largely successful, it would be wise for the US to maintain them.

During her visit, Pelosi is likely to reaffirm the US’ high regard for Taiwan’s democracy and embrace the language of shared values. She has embraced Biden’s framing of international relations as a contest between democracies and autocracies. This alone will continue to exacerbate tensions. It would be a mistake, though, to signal a major policy change away from strategic ambiguity and towards strategic clarity on Taiwan’s status. Even if the US decides later to embrace a policy shift of this size, such a message should be carefully considered and communicated clearly, and not by chance.

Congress has an important role to play but President Joe Biden and his national security team should make the final decision on US policy towards Taiwan. Getting the signals right in international politics is a key part of deterrence and, especially in East Asia, deterring both China and Taiwan’s ambitions is essential. 

Increasing insecurity in the region?

Dr John Nilsson-Wright and Ben Bland

Nancy Pelosi’s visit to Taiwan has provoked mixed responses from US allies across Asia.

For Japanese policymakers, the Taiwan issue is connected to the wider issue of regional security. Fears that a military conflict over the island will inevitably draw Japanese self-defense forces into a shooting war with China – a development that is neither formally mandated under the terms of the US-Japan Mutual Security Treaty nor necessarily constitutionally sanctioned – explains the concerns in Tokyo.

While the Japanese government of Prime Minister Fumio Kishida is increasingly worried about China’s growing military presence in the East and South China Seas, Japan’s heavy trade dependence on China and the country’s economic and security vulnerabilities make it imperative to avoid any further escalation of tensions.

Given Tokyo’s non-recognition policy towards Taiwan, Japanese ties with Taipei are handled informally by politicians of the governing Liberal Democratic Party (LDP), rather than at cabinet or foreign ministry level, and in recent weeks and months there has been an increase in visits by cross party delegations from Japan.

The death of former Prime Minister Shinzo Abe as removed from public life a vocal advocate in support of enhanced ties between Tokyo and Taipei, but with public opinion in Japan increasingly tilting in an anti-Chinese direction, and with younger politicians favouring a more combative approach towards Beijing, there is a risk that the government will face pressure at home to toughen its language on Taiwan. Bolstering deterrence through increased military cooperation among allies, along with a graduated increase in defence spending, is the best way of limiting risk over Taiwan.

Nevertheless, privately, many officials in Tokyo are likely to have viewed the Pelosi visit as an unhelpful intervention and will be puzzled and perhaps frustrated by the apparent inability of the Biden administration to persuade the US Speaker of the House of Representatives to cancel her visit.

In South Korea, the government of President Yoon Suk-yeol, faces similar pressures to Japan, given the heavy dependence of the South Korean economy on China for trade and investment opportunities.

Pelosi’s visit to the region will strikingly not include meetings with either the president or Foreign Minister Park Jin. With Yoon on vacation and Park attending the ASEAN Regional Forum meeting in Cambodia, the absence of high profile engagements for Pelosi might seem to be a purely practical matter, but Seoul may also be seeking to avoid antagonizing Beijing at a time when the Chinese government is seeking to pressure South Korea not to enhance alliance coordination with the United States and Japan or to expand its commitment to the controversial Terminal High Altitude Area Defence (THAAD) missile defence system.

As in Japan, public opinion in South Korea is increasingly anti-Chinese, but the logic of regional economic and security uncertainty, requires the Yoon government to avoid getting trapped in a worsening stand-off with Beijing.

Lars Jønson
May 1, 2007; 6:798-811
Research

Yi Zhang
Sep 1, 2005; 4:1240-1250
Research

Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens.

How Beijing is closing surveillance gaps in the South China Sea The World Today iallan.drupal 15 October 2024

The discovery of a new radar system on China’s Triton Island military base shows that Beijing is rapidly developing its intelligence capacities in contested waters, write John Pollock and Damien Symon.

New satellite images reveal how the Chinese military is dramatically expanding its capabilities on Triton Island, which looks set to become a one of Beijing’s key signal intelligence bases in the South China Sea.

Once completed, the radar system would significantly increase China’s signals intercept and electronic warfare capabilities across the disputed Paracel Islands archipelago and add to a wider surveillance network spanning much of the South China Sea.

The enhanced facility on Triton is likely to offer a challenge to China’s competitors in the region and internationally.

China seized control of the Paracels from Vietnam in a 1974 naval battle, and competition for access to it waters has intensified since the recent discovery of oil and gas reserves. Chinese and Vietnamese maritime militia clashed off the coast of Triton in 2014.

In addition, American, British and Australian naval forces have for the past decade patrolled the waters to collectively challenge China’s contested ‘nine-dash-line’ claim to large stretches of the South China Sea. 

Overlapping anti-stealth network

A year after work was first identified on Triton, satellite images from Maxar have helped build a clearer picture of Beijing’s efforts to defend this strategic waterway.

The most striking development is the construction of a new radar system, known as SIAR – synthetic impulse and aperture radar – which purportedly detects stealth aircraft. The counter-stealth radar on Triton is characterized by its distinctive octagonal structure, which resembles another SIAR system built by China on Subi Reef in the Spratly Islands, south of the Paracels, in 2017. A half-completed tower also sits near the SIAR radar on Triton, which is likely to be the operations centre.

Once completed, the radar on Triton will form what is believed to be a wider network of at least three overlapping counter-stealth radars built across Chinese bases in the South China Sea over the past decade, including on Hainan Island, home to several Chinese naval bases. 

The positioning of the radar on Triton, 320km south of Hainan, is telling, says J. Michael Dahm, Senior Resident Fellow for Aerospace and China Studies at the Mitchell Institute. 

‘SIAR radars cannot see over the curve of the Earth, which means there is a gap in China’s air surveillance coverage between Subi Reef and Hainan Island. The Triton Island site will help close that gap’, said Dahm.

The aim, he suggests, is to give China contiguous counter-stealth radar coverage of the South China Sea.

More construction underway

The satellite images reveal other building projects on Triton. One is a large pad at the end of the road network which will probably be used as a launching point for a mobile anti-ship missile battery. The building at the northeast end of the road is probably a storage building for missile transport vehicles. 

Diminishing Vietnam’s options

The development of a new counter-stealth radar system and other suspected signals intercept structures on Triton represents a notable increase in China’s intelligence capabilities in the Paracels. The Chinese Communist Party has not disclosed the purpose of the building work on Triton, but its effects on regional and global competitors are likely to be wide ranging.

For Vietnam, which is rapidly expanding its own bases in the South China Sea, the intelligence structures on Triton would significantly diminish its capacity to operate undetected in the area. Alongside existing radar on Triton which can detect sea-going vessels, Beijing now has the potential to track Vietnamese air movements and gain forewarning of Hanoi’s manoeuvres in the area, including efforts to access oil and gas deposits.

The desire to strengthen control over these resources may explain why China is fortifying Triton Island, says Bill Hayton, Associate Fellow in the Asia-Pacific Programme, Chatham House. ‘The developments might be a warning that China is planning to mount another drilling expedition’, he suggests.

Mohamad Navab
Jun 1, 2004; 45:993-1007
Thematic Reviews

Linda J. Pike
Jul 1, 2006; 47:1597-1598
Report

Weerapan Khovidhunkit
Jul 1, 2004; 45:1169-1196
Thematic Reviews

RK Tangirala
Nov 1, 1995; 36:2320-2328
Articles

Expert

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3'-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5'-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Tsz-Kiu Aaron Chow and Yangyang Li
Trans. Amer. Math. Soc. 377 (), 5993-6020.
Abstract, references and article information

Simone Cecchini and Rudolf Zeidler
Trans. Amer. Math. Soc. 377 (), 5271-5288.
Abstract, references and article information

Luiz Ricardo Abreu Melo
Proc. Amer. Math. Soc. 152 (), 5373-5380.
Abstract, references and article information

Nigy Boy's management team was forced to put out a scam alert on Thursday as news surfaced that a promoter in the Turks and Caicos Islands, inadvertently wired thousands of US dollars to who he believed was the artiste's booking team as a deposit...

Carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18–24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1–3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure–function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1–3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1–3Gal revealed the parallel β-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

1 July 2020

17 July 2020

12 August 2020

Visual Abstract

Kailun Fang
Nov 30, 2020; 0:RA120.002081v1-mcp.RA120.002081
Research

Leandro Xavier Neves
Nov 11, 2020; 0:RA120.002227v1-mcp.RA120.002227
Research

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young's modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young's modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1β secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Jiayan Guo
Dec 1, 2020; 61:1764-1775
Research Articles

Daphne E.C. Boer
Dec 23, 2020; 0:jlr.RA120001043v1-jlr.RA120001043
Research Articles

Douglas A. Andres
Dec 1, 2020; 61:1537-1537
Images in Lipid Research

Nicholas D. LeBlond
Dec 1, 2020; 61:1697-1706
Research Articles

Astrid M. Moerman
Dec 23, 2020; 0:jlr.RA120000974v1-jlr.RA120000974
Research Articles

Thibaut Bourgeois
Dec 11, 2020; 0:jlr.RA120000737v1-jlr.RA120000737
Research Articles

Abudukadier Abulizi
Dec 1, 2020; 61:1565-1576
Research Articles

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.

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The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (PIs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of PI kinases and PI phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule phosphatidylinositol. PI signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane binding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIs in general, in this review, we discuss recent studies and advances in PI lipid signaling in the retina. We specifically focus on PI lipids from vertebrate (e.g. bovine, rat, mice, toad, and zebrafish) and invertebrate (e.g. drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIs revealed from animal models and human diseases, and methods to study PI levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PI-modifying enzymes/phosphatases and further unravel PI regulation and function in the different cell types of the retina.

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3’s role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KOMac). We observed that partial Lpcat3 deficiency (approx. 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KOMac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr-/-) mice. Lpcat3KOMac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation.