Towards just transition in Africa: Green financing for urban energy solutions and job creation 9 June 2022 — 7:30AM TO 11:00AM Anonymous (not verified) 18 May 2022 Nairobi and online

This event explores the major openings and potential impediments to the development of a just transition policy in Africa.

Global climate policies towards a ‘just transition’ under the Paris Agreement should also align with and support African states’ national sustainable development priorities. In particular, the need for decent and fair job creation and the establishment of sufficient, resilient and sustainable power supply, accessible to all, and efficient energy use.

Achieving green growth requires innovative and more accessible financing models, especially as wealthy nations’ financial pledges have fallen short. Ahead of the ‘African COP27’ set to take place in Egypt in November 2022, there is a need for transformational strategic thinking and context-specific action from African governments, civil society, businesses and financiers in their green financing demands and national implementation plans.

Sustainable urban energy solutions represent a critical zone of opportunity for the development of new and more reliable green finance pathways. Africa’s rapidly expanding cities present a significant economic opportunity and source of growth. However, urban centres are also where income and energy inequalities are at their starkest. The acceleration of sustainable energy generation and use could have a transformative impact on SMEs and livelihoods across value chains.

At this event, participants will discuss the major openings and potential impediments to the development of a credible ‘just transition’ policy in Africa towards net zero goals, with a particular focus on establishing and enhancing links between green financing innovation, employment creation, sustainable power supply and generation, and sustainable energy usage and consumption in an urban environment.

This event is held in partnership with the Pan African Climate Justice Alliance (PACJA). It is part of a series on Towards Just Transition: Connecting Green Financing and Sustainable Job Creation in Africa, supported by the Chatham House Sustainability Accelerator.

This event will be held in English and French with simultaneous interpretation.

Towards just transition in Africa: Green financing for nature-based solutions and rural resilience 21 July 2022 — 9:30AM TO 1:00PM Anonymous (not verified) 30 June 2022 Libreville and online

This hybrid event in Libreville explores just transition policy and green financing for nature-based solutions, with a particular focus on the integration of job creation priorities in conservation and rural resilience.

Global climate policies towards a ‘just transition’ under the Paris Agreement should align with and support African states’ national sustainable development priorities – in particular, the need for decent and fair job creation, as well as resilient and sustainable land, environment, and ecosystem management policies.

Achieving green growth requires innovative and more accessible financing models, especially as wealthy nations’ financial pledges have fallen short. Ahead of the ‘African COP27’ set to take place in Egypt in November 2022, there is a need for transformational strategic thinking and context-specific action from African governments, civil society, businesses and financiers, in their green financing demands and national implementation plans.

Preservation of biodiversity and nature is not only critical in the global fight against climate change but is also vital for conservation-based economic development. Natural capital stocks, such as terrestrial and marine ecosystems and biodiversity, produce benefits that support societal and individual well-being and economic prosperity, such as clean air, fresh water, regulation of water flows and pollination of crops – while also acting as important carbon sinks. Financing environmental protection must go beyond compensation and contribute to creating fair social and economic conditions for incentivizing conservation.

At this hybrid event in Libreville, participants will discuss green financing for nature-based solutions, particularly the integration of plans for job creation in conservation and rural resilience within just transition planning.

This event is part of a series on Towards Just Transition: Connecting Green Financing and Sustainable Job Creation in Africa, supported by the Chatham House Sustainability Accelerator.

This event will be held in French and English with simultaneous interpretation.

This event will also be broadcast live on the Chatham House Africa Programme’s Facebook page.

Towards democracy in Sudan: Reflections on the transitional period 20 September 2022 — 2:00PM TO 4:00PM Anonymous (not verified) 31 August 2022 Online

In this webinar, panellists discuss the key outcomes of the evaluation workshop and reflect on how the experience of the transition to date should inform the realization of Sudan’s democratic aspirations going forward.

The military coup on 25 October 2021 halted Sudan’s transition to democracy and prospects for sustainable peace. Since then, members of the former regime have regained political influence, with many reinstated to senior positions.

The coup has provoked a strong reaction from the country’s pro-democracy movement and youth-led resistance committees who have led continuous peaceful protests demanding civilian democratic transition, despite a brutal crackdown by state security forces.

Those supportive of the coup have blamed the Forces of Freedom and Change (FFC), a wide coalition of pro-democracy political and civilian groups, for the mistakes of the transitional government.

However, the country’s political, security and economic situation has sharply deteriorated since the military’s action, and the progress achieved by the transitional government has been reversed, leading to an accelerating economic crisis, increased food insecurity and political instability.

The FFC, which played a key role in appointing the transitional government, has acknowledged its errors. In July 2022, it held a workshop in Khartoum, which was broadcast on social media, to evaluate its performance and identify lessons learned, in discussion with civil society actors and activists.

In this webinar, leading pro-democracy movement figures and independent experts discuss the key outcomes of the evaluation workshop and reflect on how the experience of the transition to date should inform the realization of Sudan’s democratic aspirations going forward.

This event is part of a Chatham House Africa programme project on supporting Sudan’s civilian-led democratic transition.

Towards just transition in Africa: Continental coordination on green financing and job creation 6 October 2022 — 7:00AM TO 3:30PM Anonymous (not verified) 8 September 2022 Addis Ababa and online

At this hybrid conference in Addis Ababa, speakers take stock of preparations ahead of the ‘African COP27’ in November and discuss the key priorities for streamlining continental cooperation on policy approaches to just transition.

At this hybrid conference in Addis Ababa, speakers will take stock of policy efforts and preparations ahead of the ‘African COP27’ in November and discuss the key priorities for streamlining continental cooperation on policy approaches to just transition.

Global climate policies towards a ‘just transition’ under the Paris Agreement should align with and support African states’ national sustainable development priorities – in particular, the need for decent and fair job creation, as well as resilient and sustainable land, environment and ecosystem management policies.

They must also be cognizant of African nations’ urgent requirements for sustainable and accessible energy to underpin economic development. Achieving green growth requires innovative and more accessible financing models, especially as wealthy nations’ financial pledges have fallen short. It also requires clarity and cooperation to unlock investment in both renewable and transitional energy.

African countries face collective climate and employment-related challenges. However, policymaking often remains regionally siloed according to differing political, energy sector and ecological realities. There is a need for transformational strategic thinking and context-specific action from African governments, civil society, businesses and financiers, in their green financing demands and national implementation plans.

At this hybrid conference in Addis Ababa, speakers will take stock of policy efforts and preparations ahead of the ‘African COP27’ in November and discuss the key priorities for streamlining continental cooperation on policy approaches to just transition, job creation and green financing.

This event is the third in a series on Towards just transition: Connecting green financing and sustainable job creation in Africa, supported by the Chatham House Sustainability Accelerator.

Sudan’s gold boom: Connections to conflict and transnational impacts 7 December 2022 — 2:00PM TO 3:30PM Anonymous (not verified) 24 November 2022 Online

At this event, experts will discuss Sudan’s gold sector, its connections to conflict, and transnational impacts. 

At this webinar panellists will discuss Sudan’s gold sector, its connections to conflict, and transnational impacts.

Sudan is one of the largest gold producers on the continent, with the industry constituting Sudan’s foremost source of hard currency since the secession of South Sudan in 2011 and resulting loss of oilfields.

The gold rush that has ensued has had important implications for domestic and transnational conflict dynamics. Military actors and armed groups have sought control of gold-producing areas in the peripheries and to capitalize on the flow of labour migrants, against a wider backdrop of conflict partly stemming from contestation for control between central and local actors.

International interests are prominent, including increased Russian involvement in the sector, while gold smuggling has also interlaced with mercenary activity in neighbouring CAR, Chad and Libya.
 
At this event, panellists will discuss Sudan’s gold trade, its connections to conflict, and transnational impacts, including the international politics of Sudan’s gold extraction and role of armed groups. It will also explore the environmental and socio-economic dimensions of gold in Sudan’s border areas. 
 
This roundtable is an output of the Cross-Border Conflict: Evidence, Policy and Trends (XCEPT) research programme, funded by UK Aid from the UK government.
 

Towards just transition in Africa Interview Video NCapeling 23 December 2022

Highlighting key interventions from African policymakers, business leaders, researchers, and civil society voices on green financing and implementation plans.

African countries face collective climate and employment-related challenges. However, policymaking often remains regionally siloed according to differing political, energy sector, and ecological realities.

There is a need for transformational strategic thinking and context-specific action from African governments, civil society, businesses, and financiers, in their green financing demands and national implementation plans.

This video highlights key interventions from policymakers, business leaders, researchers, and civil society voices at a series of events hosted by the Chatham House Africa programme in Nairobi, Libreville, and Addis Ababa in the lead-up to COP27.

The events series, Towards just transition: Connecting green financing and sustainable job creation in Africa, was supported by the Chatham House Sustainability Accelerator and the United Nations Development Programme.

Africa Aware: Towards just transition in Africa Audio NCapeling 1 March 2023

African countries face collective climate and job creation-related challenges, yet policymaking often remains regionally siloed.

This podcast reflects on the key policy messages from the Africa programme’s series on Towards just transition: Connecting green financing and sustainable job creation in Africa.

African countries face collective climate and job creation-related challenges. Yet policymaking often remains regionally siloed according to differing political, energy sector and ecological realities.

This output is part of a stream of work supported by the Chatham House Sustainability Accelerator and the United Nations Development Programme (UNDP). 

A critical juncture for Sudan’s democratic transition Expert comment LJefferson 28 March 2023

International pressure is essential to reach an agreement that establishes a credible civilian government.

The signing of the Framework Agreement (FA) on 5 December 2022 between Sudan’s military leaders and its leading pro-democracy parties is a major step to reversing the damage done by the disastrous military coup in October 2021.

The FA removes any formal role for the military in Sudan’s politics. A civilian head of state and prime minister will select the cabinet and chair the Defence and Security Council. The armed forces will be prohibited from non-military business activities and security sector reform will lead to a unified, professional and non-partisan national army. Elections are due to take place at the end of a two-year transitional period. 

Signatories included General Abdel Fatah Al Burhan, chair of the Sovereign Council and head of the Sudanese Armed Forces (SAF), General Mohamed Hamdan Dagalo (known as Hemedti), his deputy and Commander of the paramilitary Rapid Support Forces (RSF) and more than 40 civilian entities, including the Forces of Freedom and Change- Central Council (FFC-CC), a few other political parties, former armed movements, civil society organizations and professional associations. However, the agreement has faced criticism from the street for not being sufficiently radical, has been overshadowed at times by heightened tension between the two military leaders, and has seen sabotage attempts by supporters of the Bashir regime.

Building consensus on the Framework Agreement

The agreement meets most of the demands of the anti-coup camp, at least on paper. Yet doubts persist as to whether the military are genuine about handing over power, particularly among the neighbourhood-based resistance committees – the heart of the youth-led mobilization that forced the military to recognize the failure of their power grab. Peaceful protests against the coup have seen 125 killed and over 8,000 injured by government security forces. Many want to see Burhan and Hemedti held accountable.

Recognizing the need to expand popular support, FFC-CC leaders have been reaching out to other pro-democracy forces to build a united civilian front. They report increased buy-in from some resistance committees in the last few months, recognizing that street protests alone were not sufficient to overthrow the coup, and that engagement with the military is necessary to find a way out of the impasse.

The FA offers the only currently available path to embedding civilian politics in Sudan and has received active diplomatic support from UNITAMS, AU and IGAD (who form the Tripartite Mechanism), the Troika of the US, UK and Norway, alongside the EU, as well as Saudi Arabia and the UAE (who are members of ‘the Quad’ with the US and UK).

Broader public participation has also been developed through a series of conferences, facilitated by the Tripartite Mechanism, on five contentious issues – dismantling the old regime, the Juba Peace Agreement, Eastern Sudan, Transitional Justice and Security Sector Reform. Recommendations will be incorporated in a final political agreement. 

The political process has been overshadowed by increasingly visible tension between Burhan and Hemedti, seen in parallel foreign visits, conflicting public statements, and a heavy military presence in Khartoum. But concerns that SAF and the RSF were heading towards confrontation appear to have been assuaged thanks to international pressure and preliminary agreements reached between military and civilian signatories of the FA on security sector reform and integration. In a significant breakthrough, both sides have now agreed to draft the final agreement and transitional constitution, with the aim of forming a civilian government by 11 April.  

Potential spoilers and interests from Sudan’s regions

Progress has been made, but significant challenges remain, notably from supporters of the former Bashir regime in ‘the deep state’ and from Sudan’s historically marginalized peripheries. Old regime elements have been intensifying social media campaigns to derail the agreement and drive a wedge between the SAF and RSF, and have been accused of deliberately inciting instability in the peripheries to undermine the democratic transition. 

The Popular Defence Forces, established by the National Islamic Front in the 1990s, have been reactivated under different names in several parts of the country and there are reports of mobilization and recruitment of armed militias in Darfur. The recent public appearance of Ali Karti, the Secretary-General of the Islamic Movement, who has close relations with Islamists in SAF, has also caused renewed concern.

Two Darfuri armed movement leaders who signed the October 2020 Juba Peace Agreement (JPA) and are members of the current military-led government, have not signed the FA, allegedly due to concerns about their representation in the next government.

Despite intensive efforts to bring them on board, there is continuing disagreement over the inclusion of other members of ‘the Democratic Bloc’, a political alliance backed by Sudan’s influential neighbour Egypt, which is reportedly angry at being excluded from the Quad. The FFC-CC say that the door is open for the two Darfuri leaders and some other political parties, but they will not allow the agreement to be ‘diluted’ with political forces who intend to torpedo the transition, including by imposing a weak prime minister.

Both Burhan and Hemedti have courted support from the regions. Burhan used the 2020 SAF takeover of Al Fashaga in the contested eastern border region with Ethiopia to boost his national standing and secure backing from local tribal leaders; while Hemedti has sought to position himself as a champion of the peripheries, particularly in his Darfur heartlands, while simultaneously advancing his business interests.

Competition between them in building domestic powerbases, as well as alliances with neighbouring states, risks reigniting tensions, particularly given deep grievances and contrasting ambitions between and among Sudan’s diverse regional leaders and communities.

Visual Abstract

Visual Abstract

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

Daniel J. Geiszler
Dec 1, 2020; 0:TIR120.002216v1-mcp.TIR120.002216
Technological Innovation and Resources

Adult progenitor cell populations typically exist in a quiescent state within a controlled niche environment. However, various stresses or forms of damage can disrupt this state, which often leads to dysfunction and aging. We built a glucocorticoid (GC)-induced liver damage model of mice, found that GC stress induced liver damage, leading to consequences for progenitor cells expansion. However, the mechanisms by which niche factors cause progenitor cells proliferation are largely unknown. We demonstrate that, within the liver progenitor cells niche, Galectin-3 (Gal-3) is responsible for driving a subset of progenitor cells to break quiescence. We show that GC stress causes aging of the niche, which induces the up-regulation of Gal-3. The increased Gal-3 population increasingly interacts with the progenitor cell marker CD133, which triggers focal adhesion kinase (FAK)/AMP-activated kinase (AMPK) signaling. This results in the loss of quiescence and leads to the eventual stemness exhaustion of progenitor cells. Conversely, blocking Gal-3 with the inhibitor TD139 prevents the loss of stemness and improves liver function. These experiments identify a stress-dependent change in progenitor cell niche that directly influence liver progenitor cell quiescence and function.

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2–AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)–dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, β-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, β-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary β-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

Acute lung injury (ALI), is a rapidly progressing heterogenous pulmonary disorder that possesses a high risk of mortality. Accumulating evidence has implicated the activation of the p65 subunit of NF-κB [NF-κB(p65)] activation in the pathological process of ALI. microRNAs (miRNAs), a group of small RNA molecules, have emerged as major governors due to their post-transcriptional regulation of gene expression in a wide array of pathological processes, including ALI. The dysregulation of miRNAs and NF-κB activation has been implicated in human diseases. In the current study, we set out to decipher the convergence of miR-99b and p65 NF-κB activation in ALI pathology. We measured the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) in bronchoalveolar lavage fluid using ELISA. MH-S cells were cultured and their viability were detected with cell counting kit 8 (CCK8) assays. The results showed that miR-99b was up-regulated, while PRDM1 was down-regulated in a lipopolysaccharide (LPS)-induced murine model of ALI. Mechanistic investigations showed that NF-κB(p65) was enriched at the miR-99b promoter region, and further promoted its transcriptional activity. Furthermore, miR-99b targeted PRDM1 by binding to its 3'UTR, causing its down-regulation. This in-creased lung injury, as evidenced by increased wet/dry ratio of mouse lung, myeloperoxidase activity and pro-inflammatory cytokine secretion, and enhanced infiltration of inflammatory cells in lung tissues. Together, our findings indicate that NF-κB(p65) promotion of miR-99b can aggravate ALI in mice by down-regulating the expression of PRDM1.

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

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.

Stephanie E. Hood
Dec 1, 2020; 61:1720-1732
Research Articles

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

Ryunosuke Ohkawa
Dec 1, 2020; 61:1577-1588
Research Articles

Genta Kakiyama
Dec 1, 2020; 61:1629-1644
Research Articles

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

Aloïs Dusuel
Dec 9, 2020; 0:jlr.RA120000704v1-jlr.RA120000704
Research Articles

In SAS Customer Intelligence Studio,  when you add  Reply- node variable values in the Define Value dialog box, you might notice that two identically labeled data-grid variables are

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

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.

Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as acceptor for subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced β-glucosidase activity were similarly impaired in β-xylosidase, transglucosidase and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from Gaucher disease patients. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyse formation of XylCer. Thus, food-derived β-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.

Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition.

Nonsmall cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. While mutations in Kras and overexpression of Myc are commonly found in patients, the role of altered lipid metabolism in lung cancer and its interplay with oncogenic Myc is poorly understood. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc combined with surface analysis lipid profiling of lung tumors and transcriptomics to study the effect of Myc activity on cholesterol homeostasis. Our findings reveal that the activation of Myc leads to the accumulation of cholesteryl esters (CEs) stored in lipid droplets. Subsequent Myc deactivation leads to further increases in CEs, in contrast to tumors in which Myc was never activated. Gene expression analysis linked cholesterol transport and storage pathways to Myc activity. Our results suggest that increased Myc activity is associated with increased cholesterol influx, reduced efflux, and accumulation of CE-rich lipid droplets in lung tumors. Targeting cholesterol homeostasis is proposed as a promising avenue to explore for novel treatments of lung cancer, with diagnostic and stratification potential in human NSCLC.

Lipins are eukaryotic proteins with functions in lipid synthesis and the homeostatic control of energy balance. They execute these functions by acting as phosphatidate phosphatase enzymes in the cytoplasm and by changing gene expression after translocation into the cell nucleus, in particular under fasting conditions. Here, we asked whether nuclear translocation and the enzymatic activity of Drosophila Lipin serve essential functions and how gene expression changes, under both fed and fasting conditions, when nuclear translocation is impaired. To address these questions, we created a Lipin null mutant, a mutant expressing Lipin lacking a nuclear localization signal (Lipin^NLS), and a mutant expressing enzymatically dead Lipin. Our data support the conclusion that the enzymatic but not nuclear gene regulatory activity of Lipin is essential for survival. Notably, adult Lipin^NLS flies were not only viable but also exhibited improved life expectancy. In contrast, they were highly susceptible to starvation. Both the improved life expectancy in the fed state and the decreased survival in the fasting state correlated with changes in metabolic gene expression. Moreover, increased life expectancy of fed flies was associated with a decreased metabolic rate. Interestingly, in addition to metabolic genes, genes involved in feeding behavior and the immune response were misregulated in Lipin^NLS flies. Altogether, our data suggest that the nuclear activity of Lipin influences the genomic response to nutrient availability with effects on life expectancy and starvation resistance. Thus, nutritional or therapeutic approaches that aim at lowering nuclear translocation of lipins in humans may be worth exploring.

NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^–/– mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

The organic anion transporters (OATs) and organic anion–transporting polypeptides (OATPs) belong to the solute carrier (SLC) transporter superfamily and play important roles in handling various endogenous and exogenous compounds of anionic charge. The OATs and OATPs are often implicated in drug therapy by impacting the pharmacokinetics of clinically important drugs and, thereby, drug exposure in the target organs or cells. Various mechanisms (e.g. genetic, environmental, and disease-related factors, drug-drug interactions, and food-drug interactions) can lead to variations in the expression and activity of the anion drug-transporting proteins of OATs and OATPs, possibly impacting the therapeutic outcomes. Previous investigations mainly focused on the regulation at the transcriptional level and drug-drug interactions as competing substrates or inhibitors. Recently, evidence has accumulated that cellular trafficking, post-translational modification, and degradation mechanisms serve as another important layer for the mechanisms underlying the variations in the OATs and OATPs. This review will provide a brief overview of the major OATs and OATPs implicated in drug therapy and summarize recent progress in our understanding of the post-translational modifications, in particular ubiquitination and degradation pathways of the individual OATs and OATPs implicated in drug therapy.

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site–associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.

Glutathionylation is an important posttranslational modification that protects proteins from further oxidative damage as well as influencing protein structure and activity. In the present study, we demonstrate that the cysteine-42 residue in protein arginine N-methyltransferase 5 (PRMT5) is glutathionylated in aged mice or in cells that have been exposed to oxidative stress. Deglutathionylation of this protein is catalyzed by glutaredoxin-1 (Grx1). Using mutagenesis and subsequent biochemical analyses, we show that glutathionylation decreased the binding affinity of PRMT5 with methylosome protein-50 (MEP50) and reduced the methyltransferase activity of PRMT5. Furthermore, overexpression of PRMT5-C42A mutant caused a significant increase in histone methylation in HEK293T and A549 cells and promoted cell growth, whereas overexpression of the PRMT5-C42D mutant, a mimic of glutathionylated PRMT5, inhibited cell proliferation. Taken together, our results demonstrate a new mechanism of regulation of PRMT5 methyltransferases activity and suggest that PRMT5 glutathionylation is partly responsible for reactive oxygen species-mediated cell growth inhibition.

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.

Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

This article provides an introduction to Fourier transform-based mass spectrometry (FTMS). The key performance characteristics of FTMS, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of FTMS technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for his/her application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.

The histidine kinase Hik33 plays important roles in mediating cyanobacterial response to divergent types of abiotic stresses including cold, salt, high light (HL), and osmotic stresses. However, how these functions are regulated by Hik33 remains to be addressed. Using a hik33-deficient strain (hik33) of Synechocystis sp. PCC 6803 (Synechocystis) and quantitative proteomics, we found that Hik33 depletion induces differential protein expression highly similar to that induced by divergent types of stresses. This typically includes downregulation of proteins in photosynthesis and carbon assimilation that are necessary for cell propagation, and upregulation of heat shock proteins, chaperons, and proteases that are important for cell survival. This observation indicates that depletion of Hik33 alone mimics divergent types of abiotic stresses, and that Hik33 could be important for preventing abnormal stress response in the normal condition. Moreover, we found the majority of proteins of plasmid origin were significantly upregulated in hik33, though their biological significance remains to be addressed. Together, the systematically characterized Hik33-regulated cyanobacterial proteome, which is largely involved in stress responses, builds the molecular basis for Hik33 as a general regulator of stress responses.

CD4+ T cell responses are crucial for inducing and maintaining effective anti-cancer immunity, and the identification of human leukocyte antigen class II (HLA-II) cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed and sensitive mass spectrometry based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17 and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared to interferon gamma (IFN) treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor-antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but also such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock which has seen a rapid rise in prevalence throughout Western Europe in recent years. Following ingestion of metacercariae (parasite cysts) by the mammalian host, newly-excysted juveniles (NEJs) emerge and invade the duodenal submucosa which causes significant pathology in heavy infections. The immature larvae then migrate upwards, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients and to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognise antigens from other helminths commonly found as co-infections with rumen fluke.

Open searching has proven to be an effective strategy for identifying both known and unknown modifications in shotgun proteomics experiments. Rather than being limited to a small set of user-specified modifications, open searches identify peptides with any mass shift that may correspond to a single modification or a combination of several modifications. Here we present PTM-Shepherd, a bioinformatics tool that automates characterization of PTM profiles detected in open searches based on attributes such as amino acid localization, fragmentation spectra similarity, retention time shifts, and relative modification rates. PTM-Shepherd can also perform multi-experiment comparisons for studying changes in modification profiles, e.g. in data generated in different laboratories or under different conditions. We demonstrate how PTM-Shepherd improves the analysis of data from formalin-fixed paraffin-embedded samples, detects extreme underalkylation of cysteine in some datasets, discovers an artefactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multi-center proteomics profiling study.