Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.

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.

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.

Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32–RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, whereas the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.

16 July 2020

14 August 2020

17 September 2020

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.

A booming tech sector can unleash pan-African trade The World Today mhiggins.drupal 31 July 2022

The new African Continental Free Trade Area must embrace hyperscale data centres, cross-border digital payments and other innovations to realise its potential.

The Africa Continental Free Trade Area (AfCFTA) not only lays the groundwork for a single market across the continent, it can act as a driving force to unleash the full potential of the technology revolution that is under way across the African continent. 

To help achieve this, the AfCFTA must go beyond simply lowering barriers to the movement of goods and services, to what the World Bank calls an ‘FDI [foreign direct investment] deep scenario’. This requires harmonizing policies on investment, competition, intellectual property rights and e-commerce to encourage FDI at a greater scale. 

The World Bank estimates that the AfCFTA could increase income across the continent by 7 per cent by 2035 (an additional $445 billion), mainly by boosting intra-regional trade in manufactured goods and lifting approximately 40 million people from extreme poverty. Under an FDI deep scenario, the projected income growth jumps to 9 per cent by 2035, supporting 50 million people out of extreme poverty. 

The initial focus of the AfCFTA is on movement of goods and services and the associated financial flows through the establishment of the Pan-African Payment and Settlement System (PAPSS), a technology that enables instant local currency payment across Africa without first converting to a hard currency. In addition, harmonizing policies and easing the movement of data could enable technology to accelerate the anticipated AfCFTA income growth.

Global venture capital is pouring in

There is no doubt the African tech industry is growing. In 2021, 681 African technology companies raised $5.2 billion in equity venture funding, up from $2 billion in 2019, according to Partech Partners’ annual Africa Tech Venture Capital report. 

It is understandable why the industry has attracted global venture capital. While tech businesses are often initially focused on meeting needs in their home markets, most have a strong desire to tap into the pan-African market, with its 1.3 billion consumers across 54 countries and a combined GDP of $3.4 trillion. This in turn should attract global venture capital to invest in Africa. 

The AfCFTA has created a framework for technology-led companies to scale across the continent in a way that will impact digital infrastructure, logistics, energy and much else. For example, Africa’s hyperscale data centre capacity would benefit from the ability to locate centres in the lowest cost jurisdiction with the best energy availability and to use that to power cloud storage across the continent.

Yet various regulatory constraints, including the desire for each state to own its population’s data on local servers, prevent that. As a result, African data centres are less competitive than those in America and China. 

Similarly, logistics and other sectors would be transformed if the information on goods in transit, such as digital customs documentation, could move easily across borders while being tracked across all 54 countries. Financial services would also benefit from the ability to pay across borders in a low-cost, frictionless way.

Fintech companies should be encouraged to build technology solutions linking to PAPSS and other initiatives to accelerate the adoption-of-use cases that PAPSS supports – such as intra-Africa instant payment, embedded finance and remittances services.

AfCFTA may also unlock mergers and acquisitions (M&A) activity among African and international firms. Technology companies are using M&A to enter new markets, as the international payments platform Stripe did when it acquired the Nigerian business Paystack, and the payments business MFS Africa did when it took over the fintech start-up Baxi. 

Governments and regulators must support innovation

Given the difficulty of a country-by-country organic growth strategy across Africa, M&A is likely to increase in various technology sectors over the next few years. With the anticipated ease of doing business that the AfCFTA could facilitate, we are likely to witness further welcome consolidation, creating larger corporates that create more jobs and increase tax revenues. 

To unlock the benefits that technology will bring, governments and regulators need to play a supportive role in encouraging innovation. They will need to ensure the appropriate consumer protections are in place without stifling creativity through regulation, inefficiencies or rent-seeking. 

At the same time, governments and regulators should not permit themselves to be held to ransom by dominant incumbents, such as banks and mobile operators in the fintech space, at the expense of stifling technology companies looking to disrupt their respective industries. 

Only then will the AfCFTA allow Africa to benefit from its tech potential. 

Risana Zitha writes this article in a personal capacity

Culture notes: Europe's broken promises to Africa The World Today mhiggins.drupal 1 August 2022

Europe’s ‘gas grab’ in Africa is just the latest abuse of its relationship with the continent, says Catherine Fieschi.

When Emmanuel Macron made one of his first visits to Africa as France’s recently elected young president in 2017, his speech at Ouagadougou University in Burkina Faso was designed to set the tone for a new relationship between his country and African countries. 

‘There no longer is a French policy for Africa,’ he said.

This was a signal away from ‘la Françafrique’, with its post-colonial accents and the propping up of regimes friendly to France, to something that was more strategic, equitable and transparent – more partnership and less tutelage. 

And Europe seemed to be following suit. In March 2020 the European Union and Africa decided that they would redefine their relationship. The European Commission unveiled its vision for a ‘comprehensive strategy with Africa’. The roadmap would give Africa significantly more say over the nature and extent of the relationship, more choice and more political agency.

But what, today, is left of these aspirations? Despite repeated statements, Europe seems to be saying one thing and doing another. 

Earlier this year, after the long-awaited 6th annual EU-African Union summit in Brussels, South African president Cyril Ramaphosa was frank when he summed up the gap between stated ambitions and the current relationship. The pandemic-weary Global South had reason to be wary. Ramaphosa laid out missed opportunities, disappointment and the low expectations that act as self-fulfilling prophecies. 

Europe’s changing focus in Africa 

From the apparent high point of the Ouagadougou speech, Macron has now turned to the Organization Internationale de la Francophonie (OIF) in Africa for geopolitical purposes. His primary aim is to combat the rise of Islamist militants and terrorism in the Sahel as well as to tackle the growing influence of China and Russia in the region. 

Russian inroads – via the security firm Wagner in Mali, for instance – have given France further cause to use the OIF to counter destabilization activities. Both the United Kingdom and France train African military in the Sahel, but now, with the end of France’s anti-insurgent Operation Barkhane in Mali, the subsequent withdrawal of French troops and the increasingly established presence of the Wagner group, the security situation in the region is expected to deteriorate dramatically and become increasingly impermeable to European interests and forces.

As for development aid, Britain’s Integrated Review of Security, Defence, Development and Foreign Policy makes no bones about the fact that Asia is now a priority over Africa.

The relationship between Africa and Britain is being transformed as a result, most obviously through the cuts in development aid, with African aid cut by 66 per cent in 2021. But the nature of the relationship, which has become both more conditional and more transactional, has also changed. 

The UK is emphasizing human rights and ‘free societies’, but also pushing for free market principles rather than the kind of state involvement that some African countries often prefer as a road to accelerated and more autonomous development. 

The future of energy exports and COP27

The issue of energy exports points to what will most likely trigger the greatest disappointment in the next few years – climate and climate finance. 

Green energy deals, like the $8.5 billion COP26 package from the EU, United States and UK to South Africa, look far more problematic now in the light of Europe’s African gas-grab. Indeed, Europe is importing as much African gas as it can after the invasion of Ukraine by Russia reduced supplies. Yet African countries are still being told to curb their own use of ‘dirty’ energy. 

As an illustration, Nigeria holds 3 per cent of the world’s gas reserves, but has barely tapped them, while 40 per cent of its output is exported to Europe. In April, Italy closed deals to buy gas from Angola and the Republic of Congo, while Germany did the same with Senegal.
 

At COP15 in Copenhagen in 2009, developed countries pledged an annual $100 billion in climate finance to developing countries for both adaptation and mitigation. But pledges have never really materialized. The aid agency Oxfam estimates that only about a third of the money has been delivered. Climate finance was again the main focus of COP26 – and dismissed by Greta Thunberg as more ‘blah, blah, blah’.

This series of repeated resets, pledges and disappointments tells a story – indeed, several stories. First and foremost, it is one of arrogance and betrayal. That much is obvious. But it is also a story about stories – about how the narratives elaborated by various European countries and leaders never amount to more than a sum of transactions. 

Climate change places Europe, and other rich nations, at a crossroads in its relationship with Africa: the former holds the wealth, but also some of the keys and threats to the transition. COP27, to be held in Egypt in November, will be the next chapter in the story. 

Africa in 2023: Continuing political and economic volatility Expert comment NCapeling 6 January 2023

Despite few African trade and financial links with Russia and Ukraine, the war in Ukraine will cause civil strife in Africa due to food and energy inflation.

Africa’s economy was recovering from the COVID-19 pandemic in 2022 when a range of internal and external shocks struck such as adverse weather conditions, a devastating locust invasion, and the Russian invasion of Ukraine – all of which worsened already rapidly-rising rates of inflation and borrowing costs.

Although the direct trade and financial linkages of Africa with Russia and Ukraine are small, the war has damaged the continent’s economies through higher commodity prices, higher food, fuel, and headline inflation.

The main impact is on the increasing likelihood of civil strife because of food and energy-fuelled inflation amid an environment of heightened political instability.

Key African economies such as South Africa and Nigeria were already stuck with low growth and many African governments have seen their debt burdens increase – some such as Ethiopia and Ghana now have dollar debt trading at distressed levels – and more countries will follow in 2023.

On average the public sector debt-to-GDP ratio of African countries stood at above 60 per cent in 2022. The era of Chinese state-backed big loans and mega-projects which started 20 years ago in Angola after the end of its civil war may be coming to an end but Chinese private sector investments on the continent will continue through its Belt and Road Initiative and dual circulation model of development.

Great and middle powers building influence

Geopolitical competition in Africa has intensified in 2022, particularly among great powers such as China, Russia, the US, and the EU but also by middle powers such as Turkey, Japan, and the Gulf states.

The sixth AU-EU summit held in Brussels in February 2022 agreed on the principles for a new partnership, although the Russian invasion of Ukraine which followed disrupted these ambitions. Japan’s pledge of $30 billion in aid for Africa at TICAD 8 in August 2022 was clearly made due to the $40 billion pledged at the China–Africa summit in November 2021.

The US also launched a new strategy to strengthen its partnership and held a second US-Africa Leaders’ summit in Washington in December, the first since 2014. Russia’s ambition has been curtailed by its invasion of Ukraine, postponing its second summit with African states to 2023.

The imposition of international sanctions complicated its trade and investments, and military support such as that provided by Russian paramilitary group Wagner focused on Mali, Libya and the Central African Republic (CAR) has been curtailed.

The strategic importance of Africa has resulted in all the UN P5 members calling on the G20 to make the African Union (AU) its 21st member in 2023 under India’s presidency.

International competition to secure Africa’s critical and strategic minerals and energy products intensified in 2022 and, in the energy sector, European countries are seeking to diversify away from Russian oil and gas with alternative supplies, such as those from Africa.

Western mining companies and commodity traders are also increasingly seeking alternative supplies from Africa. Decarbonization is becoming a driver of resource nationalism and geopolitical competition in certain African mining markets, home to large deposits of critical ‘transition minerals’ such as copper, cobalt, graphite, lithium, or nickel.

COP27 was hosted in Egypt in November and gave African leaders an opportunity to shape climate discussions by pushing priority areas such as loss and damage, stranded assets, access to climate finance, adaptation, and desertification. Climate adaptation in Africa is a key condition to preserving economic growth and maintaining social cohesion.

The Horn of Africa, particularly Somalia, is suffering from one of the worst droughts in memory. The geopolitical and geoeconomic ramifications of the war in Ukraine has directly impacted the African continent by contributing to food and cooking oil inflation and humanitarian aid delivery.

Thoughout 2022 the AU was undergoing intensive reform and it struggled to respond to the growing number of security crises across the continent. Hotspots in 2023 will be in the western Sahel and Lake Chad Basin, eastern DRC, and northern Mozambique, all of them crossing state borders.

In Mozambique, a 2019 peace deal assisted by the United Nations (UN) will see the last ex-guerrillas from Renamo demobilized in 2023 to reintegrate into civilian life – some having been recruited in 1978.

In eastern Congo, M23 – one of around 120 armed groups – resumed its conflict against the central government. After lying dormant for several years, it took up arms again in 2021 and has been leading an offensive in eastern DRC against the Congolese army.

According to the UN, Rwanda has been supporting M23, and Kenya’s parliament approved in November the deployment of about 900 soldiers to the DRC as part of a joint military force from the East African Community (EAC) bloc – DRC joined the EAC in March.

In the Horn of Africa, Ethiopia saw an uneasy ceasefire agreed between the federal government and the Tigray People’s Liberation Front (TPLF).

Islamist militant groups in Africa further expanded their territorial reach in 2022, particularly in the western Sahel where al-Qaeda and Islamic State affiliates are competing for influence and continued to make inroads.

The drawdown and exit of western forces from Mali, both the French Operation Barkane and international contributions for the UN’s MINUSMA mission there, adds new dimensions to regional security challenges.

Mali’s decision in May to withdraw from the G5 Sahel has also eroded the regional security architecture. Jihadist activity may spread further into coastal states which has resulted in international partners such as France and the UK redesigning their security assistance strategies for the region.

Coups on the increase again

Since 2020, there have been successful military coups in Burkina Faso (twice), Chad, Guinea, Mali (twice), and Sudan, and failed ones in the CAR, Djibouti, Guinea-Bissau, Madagascar, Niger, and possibly Gambia and São Tomé and Príncipe.

Three national elections illustrate the state of African democracy in 2022. In Angola’s August elections, the ruling MPLA lost its absolute majority with the opposition UNITA winning the majority in Luanda for the first time.

Jessica Y. Franco
Dec 1, 2020; 19:1936-1951
Research

Tricia Rowlison
Dec 1, 2020; 19:2090-2103
Research

Alba Simats
Dec 1, 2020; 19:1921-1935
Research

Zilu Ye
Dec 28, 2020; 0:R120.002204v1-mcp.R120.002204
Review

Charlotte A. G. H. van Gelder
Dec 1, 2020; 19:1952-1967
Research

Weixian Deng
Dec 22, 2020; 0:RA120.002411v1-mcp.RA120.002411
Research

Sean A Burnap
Dec 7, 2020; 0:RA120.002305v1-mcp.RA120.002305
Research

Xinting Zhang
Dec 8, 2020; 0:RA120.002306v1-mcp.RA120.002306
Research

Mingkun Yang
Nov 24, 2020; 0:RA120.002144v1-mcp.RA120.002144
Research

Qin Zhang
Nov 17, 2020; 0:RA120.002325v1-mcp.RA120.002325
Research

Juntuo Zhou
Nov 30, 2020; 0:RA120.002384v1-mcp.RA120.002384
Research

Ka-Won Kang
Nov 30, 2020; 0:RA120.002169v1-mcp.RA120.002169
Research

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

Matthias Schittmayer
Dec 1, 2020; 19:2104-2114
Research

Laura F Fielden
Nov 11, 2020; 0:RA120.002370v1-mcp.RA120.002370
Research

Johannes Griss
Dec 1, 2020; 19:2115-2124
Technological Innovation and Resources

Tirsa L. E. van Westering
Dec 1, 2020; 19:2047-2067
Research

Diana Samodova
Dec 1, 2020; 19:2139-2156
Technological Innovation and Resources

Philipp Emanuel Geyer
Dec 17, 2020; 0:RA120.002359v1-mcp.RA120.002359
Research

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

Montgomery Blencowe
Dec 23, 2020; 0:jlr.RA120000713v1-jlr.RA120000713
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.

Mendelian randomization (MR) of lipid traits in coronary artery disease (CAD) has provided evidence for causal associations of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) in CAD, but many lipid trait genetic variants have pleiotropic effects on other cardiovascular risk factors that may bias MR associations. The goal of this study was to evaluate pleiotropic effects of lipid trait genetic variants and to account for these effects in MR of lipid traits in CAD. We performed multivariable MR using inverse variance-weighted (IVW) and MR-Egger methods in large (n ≥ 300,000) GWAS datasets. We found that 30% of lipid trait genetic variants have effects on metabolic syndrome traits, including body mass index (BMI), type 2 diabetes (T2D), and systolic blood pressure (SBP). Nonetheless, in multivariable MR analysis, LDL-C, high-density lipoprotein cholesterol (HDL-C), TG, BMI, T2D, and SBP are independently associated with CAD, and each of these associations is robust to adjustment for directional pleiotropy. MR at loci linked to direct effects on HDL-C and TG suggests locus- and mechanism-specific causal effects of these factors on CAD.

Genome-wide association studies (GWAS) have implicated ~380 genetic loci for plasma lipid regulation. However, these loci only explain 17-27% of the trait variance and a comprehensive understanding of the molecular mechanisms has not been achieved. In this study, we utilized an integrative genomics approach leveraging diverse genomic data from human populations to investigate whether genetic variants associated with various plasma lipid traits, namely total cholesterol (TC), high and low density lipoprotein cholesterol (HDL and LDL), and triglycerides (TG), from GWAS were concentrated on specific parts of tissue-specific gene regulatory networks. In addition to the expected lipid metabolism pathways, gene subnetworks involved in ‘interferon signaling’, ‘autoimmune/immune activation’, ‘visual transduction’, and ‘protein catabolism’ were significantly associated with all lipid traits. Additionally, we detected trait-specific subnetworks, including cadherin-associated subnetworks for LDL, glutathione metabolism for HDL, valine, leucine and isoleucine biosynthesis for TC, and insulin signaling and complement pathways for TG. Finally, utilizing gene-gene relations revealed by tissue-specific gene regulatory networks, we detected both known (e.g. APOH, APOA4, and ABCA1) and novel (e.g. F2 in adipose tissue) key regulator genes in these lipid-associated subnetworks. Knockdown of the F2 gene (Coagulation Factor II, Thrombin) in 3T3-L1 and C3H10T1/2 adipocytes reduced gene expression of Abcb11, Apoa5, Apof, Fabp1, Lipc, and Cd36, reduced intracellular adipocyte lipid content, and increased extracellular lipid content, supporting a link between adipose thrombin and lipid regulation. Our results shed light on the complex mechanisms underlying lipid metabolism and highlight potential novel targets for lipid regulation and lipid-associated diseases.

Among the virulence factors in Neisseria infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-B via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS³). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4'-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.

Some cases of chylomicronemia are caused by autoantibodies against glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1), an endothelial cell protein that shuttles LPL to the capillary lumen. GPIHBP1 autoantibodies prevent binding and transport of LPL by GPIHBP1, thereby disrupting the lipolytic processing of triglyceride-rich lipoproteins. Here, we review the "GPIHBP1 autoantibody syndrome" and summarize clinical and laboratory findings in 22 patients. All patients had GPIHBP1 autoantibodies and chylomicronemia, but we did not find a correlation between triglyceride levels and autoantibody levels. Many of the patients had a history of pancreatitis, and most had clinical and/or serological evidence of autoimmune disease. IgA autoantibodies were present in all patients, and IgG4 autoantibodies were present in 19 of 22 patients. Patients with GPIHBP1 autoantibodies had low plasma LPL levels, consistent with impaired delivery of LPL into capillaries. Plasma levels of GPIHBP1, measured with a monoclonal antibody–based ELISA, were very low in 17 patients, reflecting the inability of the ELISA to detect GPIHBP1 in the presence of autoantibodies (immunoassay interference). However, GPIHBP1 levels were very high in five patients, indicating little capacity of their autoantibodies to interfere with the ELISA. Recently, several GPIHBP1 autoantibody syndrome patients were treated successfully with rituximab, resulting in the disappearance of GPIHBP1 autoantibodies and normalization of both plasma triglyceride and LPL levels. The GPIHBP1 autoantibody syndrome should be considered in any patient with newly acquired and unexplained chylomicronemia.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC–MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.

Recent efforts in gut microbiome studies have highlighted the importance of explicitly describing the ecological processes beyond correlative analysis. However, we are still at the early stage of understanding the organizational principles of the gut ecosystem, partially because of the limited information provided by currently used analytical tools in ecological modeling practices. Proteomics and metaproteomics can provide a number of insights for ecological studies, including biomass, matter and energy flow, and functional diversity. In this Mini Review, we discuss proteomics and metaproteomics-based experimental strategies that can contribute to studying the ecology, in particular at the mucosal-luminal interface (MLI) where the direct host-microbiome interaction happens. These strategies include isolation protocols for different MLI components, enrichment methods to obtain designated array of proteins, probing for specific pathways, and isotopic labeling for tracking nutrient flow. Integration of these technologies can generate spatiotemporal and site-specific biological information that supports mathematical modeling of the ecosystem at the MLI.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including actin dynamics and focal adhesion kinase (FAK) activity and one resistant cell line showed a strong migration phenotype. Pharmacological inhibition of the kinases FAK by Defactinib and of CHEK1 by Rabusertib showed a statistically significant sensitization to radiation in radioresistant PDAC cells. Together, the presented data map a comprehensive molecular network of radiation-induced signaling, improves the understanding of radioresistance and provides avenues for developing radiotherapeutic strategies.

The neuronal basis of complex social behavior is still poorly understood. In honeybees, reproductive investment decisions are made at the colony-level. Queens develop from female-destined larvae that receive alloparental care from nurse bees in the form of ad-libitum royal jelly (RJ) secretions. Typically, the number of raised new queens is limited but genetic breeding of "royal jelly bees" (RJBs) for enhanced RJ production over decades has led to a dramatic increase of reproductive investment in queens. Here, we compare RJBs to unselected Italian bees (ITBs) to investigate how their cognitive processing of larval signals in the mushroom bodies (MBs) and antennal lobes (ALs) may contribute to their behavioral differences. A cross-fostering experiment confirms that the RJB syndrome is mainly due to a shift in nurse bee alloparental care behavior. Using olfactory conditioning of the proboscis extension reflex, we show that the RJB nurses spontaneously respond more often to larval odors compared with ITB nurses but their subsequent learning occurs at similar rates. These phenotypic findings are corroborated by our demonstration that the proteome of the brain, particularly of the ALs differs between RJBs and ITBs. Notably, in the ALs of RJB newly emerged bees and nurses compared with ITBs, processes of energy and nutrient metabolism, signal transduction are up-regulated, priming the ALs for receiving and processing the brood signals from the antennae. Moreover, highly abundant major royal jelly proteins and hexamerins in RJBs compared with ITBs during early life when the nervous system still develops suggest crucial new neurobiological roles for these well-characterized proteins. Altogether, our findings reveal that RJBs have evolved a strong olfactory response to larvae, enabled by numerous neurophysiological adaptations that increase the nurse bees' alloparental care behavior.

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [³⁵S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

High-speed analysis of large (prote)omics sample sets at the rate of thousands or millions of samples per day on a single platform has been a challenge since the beginning of proteomics. For many years, ESI-based MS methods have dominated proteomics because of their high sensitivity and great depth in analyzing complex proteomes. However, despite improvements in speed, ESI-based MS methods are fundamentally limited by their sample introduction, which excludes off-line sample preparation/fractionation because of the time required to switch between individual samples/sample fractions, and therefore being dependent on the speed of on-line sample preparation methods such as liquid chromatography. Laser-based ionization methods have the advantage of moving from one sample to the next without these limitations, being mainly restricted by the speed of modern sample stages, i.e. 10 ms or less between samples. This speed matches the data acquisition speed of modern high-performing mass spectrometers whereas the pulse repetition rate of the lasers (>1 kHz) provides a sufficient number of desorption/ionization events for successful ion signal detection from each sample at the above speed of the sample stages. Other advantages of laser-based ionization methods include the generally higher tolerance to sample additives and contamination compared with ESI MS, and the contact-less and pulsed nature of the laser used for desorption, reducing the risk of cross-contamination. Furthermore, new developments in MALDI have expanded its analytical capabilities, now being able to fully exploit high-performing hybrid mass analyzers and their strengths in sensitivity and MS/MS analysis by generating an ESI-like stable yield of multiply charged analyte ions. Thus, these new developments and the intrinsically high speed of laser-based methods now provide a good basis for tackling extreme sample analysis speed in the omics.

MS-based proteome profiling has become increasingly comprehensive and quantitative, yet a persistent shortcoming has been the relatively large samples required to achieve an in-depth measurement. Such bulk samples, typically comprising thousands of cells or more, provide a population average and obscure important cellular heterogeneity. Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells, a level of coverage that required an input of thousands of cells just a few years ago. This review discusses important factors and parameters that should be optimized across the workflow for single-cell and other low-input measurements. It also highlights recent developments that have advanced the field and opportunities for further development.

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.

Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.

We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.