Subcellular Signaling Research Articles

Deciphering Protein Secretion from the Brain to Cerebrospinal Fluid for Biomarker Discovery.

Cerebrospinal fluid (CSF) is an essential matrix for the discovery of neurological disease biomarkers. However, the high dynamic range of protein concentrations in CSF hinders the detection of the least abundant protein biomarkers by untargeted mass spectrometry. It is thus beneficial to gain a deeper understanding of the secretion processes within the brain. Here, we aim to explore if and how the secretion of brain proteins to the CSF can be predicted. By combining a curated CSF proteome and the brain elevated proteome of the Human Protein Atlas, brain proteins were classified as CSF or non-CSF secreted. A machine learning model was trained on a range of sequence-based features to differentiate between CSF and non-CSF groups and effectively predict the brain origin of proteins. The classification model achieves an area under the curve of 0.89 if using high confidence CSF proteins. The most important prediction features include the subcellular localization, signal peptides, and transmembrane regions. The classifier generalized well to the larger brain detected proteome and is able to correctly predict novel CSF proteins identified by affinity proteomics. In addition to elucidating the underlying mechanisms of protein secretion, the trained classification model can support biomarker candidate selection.

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia

ABSTRACTAntibody–drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg−1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

A multiscale predictive digital twin for neurocardiac modulation.

Cardiac function is tightly regulated by the autonomic nervous system (ANS). Activation of the sympathetic nervous system increases cardiac output by increasing heart rate and stroke volume, while parasympathetic nerve stimulation instantly slows heart rate. Importantly, imbalance in autonomic control of the heart has been implicated in the development of arrhythmias and heart failure. Understanding of the mechanisms and effects of autonomic stimulation is a major challenge because synapses in different regions of the heart result in multiple changes to heart function. For example, nerve synapses on the sinoatrial node (SAN) impact pacemaking, while synapses on contractile cells alter contraction and arrhythmia vulnerability. Here, we present a multiscale neurocardiac modelling and simulator tool that predicts the effect of efferent stimulation of the sympathetic and parasympathetic branches of the ANS on the cardiac SAN and ventricular myocardium. The model includes a layered representation of the ANS and reproduces firing properties measured experimentally. Model parameters are derived from experiments and atomistic simulations. The model is a first prototype of a digital twin that is applied to make predictions across all system scales, from subcellular signalling to pacemaker frequency to tissue level responses. We predict conditions under which autonomic imbalance induces proarrhythmia and can be modified to prevent or inhibit arrhythmia. In summary, the multiscale model constitutes a predictive digital twin framework to test and guide high-throughput prediction of novel neuromodulatory therapy. KEY POINTS: A multi-layered model representation of the autonomic nervous system that includes sympathetic and parasympathetic branches, each with sparse random intralayer connectivity, synaptic dynamics and conductance based integrate-and-fire neurons generates firing patterns in close agreement with experiment. A key feature of the neurocardiac computational model is the connection between the autonomic nervous system and both pacemaker and contractile cells, where modification to pacemaker frequency drives initiation of electrical signals in the contractile cells. We utilized atomic-scale molecular dynamics simulations to predict the association and dissociation rates of noradrenaline with the β-adrenergic receptor. Multiscale predictions demonstrate how autonomic imbalance may increase proclivity to arrhythmias or be used to terminate arrhythmias. The model serves as a first step towards a digital twin for predicting neuromodulation to prevent or reduce disease.

Integrative human atrial modelling unravels interactive protein kinase A and Ca2+/calmodulin-dependent protein kinase II signalling as key determinants of atrial arrhythmogenesis.

Atrial fibrillation (AF), the most prevalent clinical arrhythmia, is associated with atrial remodelling manifesting as acute and chronic alterations in expression, function, and regulation of atrial electrophysiological and Ca2+-handling processes. These AF-induced modifications crosstalk and propagate across spatial scales creating a complex pathophysiological network, which renders AF resistant to existing pharmacotherapies that predominantly target transmembrane ion channels. Developing innovative therapeutic strategies requires a systems approach to disentangle quantitatively the pro-arrhythmic contributions of individual AF-induced alterations. Here, we built a novel computational framework for simulating electrophysiology and Ca2+-handling in human atrial cardiomyocytes and tissues, and their regulation by key upstream signalling pathways [i.e. protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)] involved in AF-pathogenesis. Populations of atrial cardiomyocyte models were constructed to determine the influence of subcellular ionic processes, signalling components, and regulatory networks on atrial arrhythmogenesis. Our results reveal a novel synergistic crosstalk between PKA and CaMKII that promotes atrial cardiomyocyte electrical instability and arrhythmogenic triggered activity. Simulations of heterogeneous tissue demonstrate that this cellular triggered activity is further amplified by CaMKII- and PKA-dependent alterations of tissue properties, further exacerbating atrial arrhythmogenesis. Our analysis reveals potential mechanisms by which the stress-associated adaptive changes turn into maladaptive pro-arrhythmic triggers at the cellular and tissue levels and identifies potential anti-AF targets. Collectively, our integrative approach is powerful and instrumental to assemble and reconcile existing knowledge into a systems network for identifying novel anti-AF targets and innovative approaches moving beyond the traditional ion channel-based strategy.

Redox signaling in impaired cascades of wound healing: promising approach.

In the aging communities, wound healing management is a quite remarkable problem especially in elderly individuals. The optimal level of healing of wounds developed spontaneously or due to surgery is of critical importance in order to prevent the negative effects that may occur due to delayed healing (for example, organ or system damage caused by infections that may develop in the wound area). The deteriorated subcellular redox signaling is considered to be as the main factor in the chronicity of wounds. The pivotal role of mitochondria in redox regulation reveals the importance of modulation of redox signaling pathways in senescent cells. Secretory factors released upon the acquisition of senescence-associated secretory phenotype (SASP) function in a paracrine manner to disseminate impaired tissue redox status by affecting the redox metabolome of nearby cells, which could promote age-related pro-inflammatory pathologies. Evaluating the wound-site redox regulation in impaired redox signaling pathways may help prevent the formation of chronic wounds and the development of long-term complications of the wounds, especially in the elderly. Using the redox modulatory pharmacologically active substances targeting the senescent cells in chronic wound areas hopefully opens a new avenue in wound management. As the signaling mechanisms of wound healing and its relationship with advanced age become more clearly understood, many promising therapeutic approaches and redox modulator substances are coming into clinical view for the management of chronic wounds.

A dynamical model of TGF-β activation in asthmatic airways.

Excessive activation of the regulatory cytokine transforming growth factor $\beta $ (TGF-$\beta $) via contraction of airway smooth muscle (ASM) is associated with the development of asthma. In this study, we develop an ordinary differential equation model that describes the change in density of the key airway wall constituents, ASM and extracellular matrix (ECM), and their interplay with subcellular signalling pathways leading to the activation of TGF-$\beta $. We identify bistable parameter regimes where there are two positive steady states, corresponding to either reduced or elevated TGF-$\beta $ concentration, with the latter leading additionally to increased ASM and ECM density. We associate the former with a healthy homeostatic state and the latter with a diseased (asthmatic) state. We demonstrate that external stimuli, inducing TGF-$\beta $ activation via ASM contraction (mimicking an asthmatic exacerbation), can perturb the system irreversibly from the healthy state to the diseased one. We show that the properties of the stimuli, such as their frequency or strength, and the clearance of surplus active TGF-$\beta $, are important in determining the long-term dynamics and the development of disease. Finally, we demonstrate the utility of this model in investigating temporal responses to bronchial thermoplasty, a therapeutic intervention in which ASM is ablated by applying thermal energy to the airway wall. The model predicts the parameter-dependent threshold damage required to obtain irreversible reduction in ASM content, suggesting that certain asthma phenotypes are more likely to benefit from this intervention.

Isoform-specific functions of Ras in T-cell development and differentiation.

Ras GTPases, well characterized for their role in oncogenesis, are the cells' molecular switches that signal to maintain immune homeostasis through cellular development, proliferation, differentiation, survival, and apoptosis. In the immune system, T cells are the central players that cause autoimmunity if dysregulated. Antigen-specific T-cell receptor (TCR) stimulation activates Ras-isoforms, which exhibit isoform-specific activator and effector requirements, functional specificities, and a selective role in T-cell development and differentiation. Recent studies show the role of Ras in T-cell-mediated autoimmune diseases; however, there is a scarcity of knowledge about the role of Ras in T-cell development and differentiation. To date, limited studies have demonstrated Ras activation in response to positive and negative selection signals and Ras isoform-specific signaling, including subcellular signaling, in immune cells. The knowledge of isoform-specific functions of Ras in T cells is essential, but still inadequate to develop the T-cell-targeted Ras isoform-specific treatment strategies for the diseases caused by altered Ras-isoform expression and activation in T cells. In this review, we discuss the role of Ras in T-cell development and differentiation, critically analyzing the isoform-specific functions.

Autonomic control of ventricular function in health and disease: current state of the art.

Cardiac autonomic dysfunction is one of the main pillars of cardiovascular pathophysiology. The purpose of this review is to provide an overview of the current state of the art on the pathological remodeling that occurs within the autonomic nervous system with cardiac injury and available neuromodulatory therapies for autonomic dysfunction in heart failure. Data from peer-reviewed publications on autonomic function in health and after cardiac injury are reviewed. The role of and evidence behind various neuromodulatory therapies both in preclinical investigation and in-use in clinical practice are summarized. A harmonic interplay between the heart and the autonomic nervous system exists at multiple levels of the neuraxis. This interplay becomes disrupted in the setting of cardiovascular disease, resulting in pathological changes at multiple levels, from subcellular cardiac signaling of neurotransmitters to extra-cardiac, extra-thoracic remodeling. The subsequent detrimental cycle of sympathovagal imbalance, characterized by sympathoexcitation and parasympathetic withdrawal, predisposes to ventricular arrhythmias, progression of heart failure, and cardiac mortality. Knowledge on the etiology and pathophysiology of this condition has increased exponentially over the past few decades, resulting in a number of different neuromodulatory approaches. However, significant knowledge gaps in both sympathetic and parasympathetic interactions and causal factors that mediate progressive sympathoexcitation and parasympathetic dysfunction remain. Although our understanding of autonomic imbalance in cardiovascular diseases has significantly increased, specific, pivotal mediators of this imbalance and the recognition and implementation of available autonomic parameters and neuromodulatory therapies are still lagging.

Utilizing flow cytometry sorting signal width to enrich for cells positive to endogenous gene integration of fluorescent proteins.

Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.

PO-02-154 CHRONIC SYMPATHETIC REMODELING IN HEART FAILURE IMPACTS WHOLE-HEART CAMP SIGNALING AND ARRHYTHMOGENESIS

Elevated sympathetic tone and b-AR desensitization are characteristics associated with heart failure (HF) and are contributors to the initiation of arrhythmia. Studies have shown that altered b-AR responsiveness in HF leads to altered compartmentalization and imparied sub-cellular cAMP signaling. Yet, if cAMP signaling is heterogeneously remodeled throughout the intact heart, and how this may contribute to arrhythmia is unknown. To assess whole-heart cAMP signaling and functional responses to adrenergic activation in HF. HF was induced via transverse aortic constriction in cardiac specific CAMPER reporter mice, expressing a FRET-based cAMP biosensor. HF was confirmed by echocardiography and hearts were Langendorff-perfused for simultaneous cAMP and Ca2+ imaging on an integrated whole-heart optical mapping FRET imaging system. Sham mice underwent the same procedure but without constriction of the aorta. Baseline cAMP levels, assessed as the FRET ratio, were uniform throughout the ventricles of male and female Sham hearts. Conversely, in HF, baseline levels of cAMP were higher in the apex vs. the base, particularly in female hearts. Low-dose b1-AR stimulation (100nM NE) led to greater cAMP response in male HF hearts vs. Sham, this response was uniform across the male heart. In female HF hearts, there was a trend toward a greater cAMP response in basal, but not apical regions. At high dose b1-AR stimulation (500nM NE), male and female HF hearts had reduced cAMP responses compared to Sham, mainly in the female apex, where dose-dependent responses were lost. Similar apex-base differences were evident following b1 and b2-AR stimulation (100-500nM Epi), with lower cAMP responses in the female apex in HF. Uniform cAMP responses in male and female Sham hearts were accompanied by uniform shortening of the Ca2+ transient duration (CaTD) in response to low dose b1-AR stimulation. Following HF, baseline CaTD was shorter than in Sham hearts and the response to b-AR stimulation was reduced. Moreover, CaTD responses were no longer spatially uniform, where apical CaTD shortened beyond that of basal regions in HF. At high dose b1-AR stimulation, ventricular arrhythmia was observed in 8/10 HF hearts, vs 0/4 Sham hearts. Using whole-heart cAMP and Ca2+ imaging, we have shown cAMP activity and responses became spatially heterogeneous in HF, mainly in female hearts. Heterogeneity in cAMP activity was associated with heterogenous Ca2+ responses and ventricular arrhythmia.

PO-455617-7 CHRONIC SYMPATHETIC REMODELING IN HEART FAILURE IMPACTS WHOLE-HEART CAMP SIGNALING AND ARRHYTHMOGENESIS.

Elevated sympathetic tone and b-AR desensitization are characteristics associated with heart failure (HF) and are contributors to the initiation of arrhythmia. Studies have shown that altered b-AR responsiveness in HF leads to altered compartmentalization and imparied sub-cellular cAMP signaling. Yet, if cAMP signaling is heterogeneously remodeled throughout the intact heart, and how this may contribute to arrhythmia is unknown. To assess whole-heart cAMP signaling and functional responses to adrenergic activation in HF. HF was induced via transverse aortic constriction in cardiac specific CAMPER reporter mice, expressing a FRET-based cAMP biosensor. HF was confirmed by echocardiography and hearts were Langendorff-perfused for simultaneous cAMP and Ca2+ imaging on an integrated whole-heart optical mapping FRET imaging system. Sham mice underwent the same procedure but without constriction of the aorta. Baseline cAMP levels, assessed as the FRET ratio, were uniform throughout the ventricles of male and female Sham hearts. Conversely, in HF, baseline levels of cAMP were higher in the apex vs. the base, particularly in female hearts. Low-dose b1-AR stimulation (100nM NE) led to greater cAMP response in male HF hearts vs. Sham, this response was uniform across the male heart. In female HF hearts, there was a trend toward a greater cAMP response in basal, but not apical regions. At high dose b1-AR stimulation (500nM NE), male and female HF hearts had reduced cAMP responses compared to Sham, mainly in the female apex, where dose-dependent responses were lost. Similar apex-base differences were evident following b1 and b2-AR stimulation (100-500nM Epi), with lower cAMP responses in the female apex in HF. Uniform cAMP responses in male and female Sham hearts were accompanied by uniform shortening of the Ca2+ transient duration (CaTD) in response to low dose b1-AR stimulation. Following HF, baseline CaTD was shorter than in Sham hearts and the response to b-AR stimulation was reduced. Moreover, CaTD responses were no longer spatially uniform, where apical CaTD shortened beyond that of basal regions in HF. At high dose b1-AR stimulation, ventricular arrhythmia was observed in 8/10 HF hearts, vs 0/4 Sham hearts. Using whole-heart cAMP and Ca2+ imaging, we have shown cAMP activity and responses became spatially heterogeneous in HF, mainly in female hearts. Heterogeneity in cAMP activity was associated with heterogenous Ca2+ responses and ventricular arrhythmia.

CD40 induces selective routing of Ras isoforms to subcellular compartments.

Ras GTPases are central to cellular signaling and oncogenesis. The three loci of the Ras gene encode for four protein isoforms namely Harvey-Ras (H-Ras), Kirsten-Ras (K-Ras 4A and 4B), and Neuroblastoma-Ras (N-Ras) which share ~ 80% sequence similarity and used to be considered functionally redundant. The small molecule inhibitors of Ras lack specificity for the isoforms leading to widespread toxicity in Ras-targeted therapeutics. Ras isoforms' tissue-specific expression and selective association with carcinogenesis, embryonic development, and infection suggested their non-redundancy. We show that CD40, an antigen-presenting cell (APC)-expressed immune receptor, induces selective relocation of H-Ras, K-Ras, and N-Ras to the Plasma membrane (PM) lipid rafts, mitochondria, endoplasmic reticulum (ER), but not to the Golgi complex (GC). The two palmitoylated Ras isoforms-H-Ras and N-Ras-have a similar pattern of colocalization into the lipid-rich raft microdomain of the PM at early time points when compared to non-palmitoylated K-Ras (4B) with polylysine residues. CD40-induced trafficking of H-Ras and K-Ras to mitochondria and ER was found to be similar but different from that of N-Ras. Trafficking of all the Ras isoforms to the GC was independent of CD40 stimulation. The receptor-driven trafficking and spatial segregation of H-Ras, K-Ras, and N-Ras imply isoform-specific subcellular signaling platforms for the functional non-redundancy of Ras isoforms. PDB structures have been modified to illustrate various signaling proteins.

The adhesion G protein-coupled receptor VLGR1/ADGRV1 controls autophagy.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest known adhesion G protein-coupled receptor. Mutations in VLGR1/ADGRV1 cause Usher syndrome (USH), the most common form of hereditary deaf-blindness, and have been additionally linked to epilepsy. Although VLGR1/ADGRV1 is almost ubiquitously expressed, little is known about the subcellular function and signalling of the VLGR1 protein and thus about mechanisms underlying the development of diseases. Using affinity proteomics, we identified key components of autophagosomes as putative interacting proteins of VLGR1. In addition, whole transcriptome sequencing of the retinae of the Vlgr1/del7TM mouse model revealed altered expression profiles of gene-related autophagy. Monitoring autophagy by immunoblotting and immunocytochemistry of the LC3 and p62 as autophagy marker proteins revealed evoked autophagy in VLGR1-deficient hTERT-RPE1 cells and USH2C patient-derived fibroblasts. Our data demonstrate the molecular and functional interaction of VLGR1 with key components of the autophagy process and point to an essential role of VLGR1 in the regulation of autophagy at internal membranes. The close association of VLGR1 with autophagy helps to explain the pathomechanisms underlying human USH and epilepsy related to VLGR1 defects.

Abstract 87: The role of miro1-mediated mitochondrial positioning in the development and metastasis of breast cancer

Abstract Miro1 is an outer mitochondrial membrane protein that links mitochondria to motor protein complexes to support subcellular mitochondrial trafficking. Preliminary observations and published work has established that metastatic breast cancer cells have increased expression of Miro1 in comparison to breast cancer cells with low metastatic potential. Furthermore, tumors from breast cancer patients with high Miro1 expression have poorer survival rates compared to patients harboring tumors with low Miro1 expression. This data suggests that Miro1 has a functional role in aggressive breast cancers although this has not been widely studied or confirmed in advanced preclinical models. The objective of our research is to investigate the role of Miro1 in breast cancer tumorigenesis and metastasis. We hypothesize that that Miro1-mediated mitochondrial dynamics support subcellular signaling events driving cell migration, invasion, and tumorigenesis. To examine the function of Miro1 in promoting metastatic phenotypes in vitro, we generated human breast cancer cell lines (MDA-MB-231, MDA-MB-468, T-47d, and MCF7) with Miro1 knockdown using adenoviral shRNA. Knockdown of Miro1 to levels greater than 50% of that in control cells resulted in mitochondria sequestered around the nucleus and reduced cell migration. Additionally, MDA-MB-231 cells with ≥50% Miro1 knockdown resulted in a significant decrease in cell invasion through Matrigel coated membranes in transwell invasion assays. Our research has also uncovered altered phosphorylation of key cell migration and stress-response proteins including vinculin, focal adhesion kinase and ERK ½ when Miro1 is knocked down. To investigate the role of Miro1 in vivo, we generated a novel transgenic mouse model with inducible, tissue specific Miro1 deletion in mammary epithelial cells with concurrent polyomavirus middle T-antigen oncogene activation. We found that when breast cancer formation is induced utilizing the transgenic middle-T antigen system, our control mice form tumors at all mammary gland sites and have metastasis to the lungs. Conversely, mice with concurrent Miro1 deletion and middle T- antigen activation fail to develop tumors. This data suggests that Miro1 has a functional role in both mammary tumor onset and tumor cell migration and invasion, providing a possible new biomarker of tumor status and pathway for therapeutic intervention. Citation Format: Randi Gravelle, Margaret McCoy, Nathaniel Shannon, Martin Chang, Brian Cunniff. The role of miro1-mediated mitochondrial positioning in the development and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 87.

Purification and biochemical analysis of native AMPA receptors from three different mammalian species.

The majority of fast, excitatory synaptic transmission in the central nervous system (CNS) is mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), which are glutamate-activated ion channels integral to synaptic plasticity, motor coordination, learning, and memory. Native AMPARs are multiprotein assemblies comprised of a tetrameric receptor core that co-assembles with a broad range of peripheral auxiliary proteins which shape subcellular localization and signaling properties of the resulting complexes. Structure determination of AMPARs has traditionally relied on recombinant expression systems; however, these methods are not well suited to elucidate the diverse array of AMPAR assemblies that are differentially expressed in mammalian brains. While recent studies of native receptor complexes have advanced our understanding of endogenous assemblies, receptors thus far have only been isolated from rodent brain tissue. Here, we employed an immunoaffinity purification strategy to isolate native AMPARs from the brains of three different mammals-pigs, sheep, and cows. Compared to rodents, pigs, sheep, and cows are ungulate mammals, animals with closer genomic identity with humans. Here we determined the molecular size, overall yield, and purity of native AMPARs isolated from these three mammals, thereby demonstrating that structural determination and biochemical analysis is possible from a clade of mammals evolutionarily distinct from rodents.

Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells.

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favored as a therapeutic target due to blunted GIPR responses in T2D patients and conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology, following the realization that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R/GIPR agonists with superior capacity for controlling blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here, we have directly compared surface expression, trafficking, and signaling characteristics of both incretin receptors in pancreatic beta cells to identify potential differences that might underlie distinct pharmacological responses associated with each receptor. Our results indicate increased cell surface levels, internalization, degradation, and endosomal vs plasma membrane activity for the GLP-1R, while the GIPR is instead associated with increased plasma membrane recycling, reduced desensitization, and enhanced downstream signal amplification. These differences might have potential implications for the capacity of each incretin receptor to control beta cell function.

A core UPS molecular complement implicates unique endocytic compartments at the parasite–host interface in Giardia lamblia

ABSTRACT Unconventional protein secretion (UPS) plays important roles in cell physiology. In contrast to canonical secretory routes, UPS does not generally require secretory signal sequences and often bypasses secretory compartments such as the ER and the Golgi apparatus. Giardia lamblia is a protist parasite with reduced subcellular complexity which releases several proteins, some of them virulence factors, without canonical secretory signals. This implicates UPS at the parasite–host interface. No dedicated machinery nor mechanism(s) for UPS in Giardia are currently known, although speculations on the involvement of endocytic organelles called PV/PECs, have been put forth. To begin to address the question of whether PV/PECs are implicated in virulence-associated UPS and to define the composition of molecular machinery involved in protein release, we employed affinity purification and mass spectrometry, coupled to microscopy-based subcellular localization and signal correlation quantification to investigate the interactomes of 11 reported unconventionally secreted proteins, all predicted to be cytosolic. A subset of these are associated with PV/PECs. Extended and validated interactomes point to a core PV/PECs-associated UPS machinery, which includes uncharacterized and Giardia-specific coiled-coil proteins and NEK kinases. Finally, a subset of the alpha-giardin protein family was enriched in all PV/PECs-associated protein interactomes, highlighting a previously unappreciated role for these proteins at PV/PECs and in UPS. Taken together, our results provide the first characterization of a virulence-associated UPS protein complex in Giardia lamblia at PV/PECs, suggesting a novel link between these primarily endocytic and feeding organelles and UPS at the parasite–host interface.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

N-glycosylation on Asn-57 is required for the correct HAI-2 protein folding and protease inhibitory activity.

Hepatocyte growth factor activator inhibitor (HAI)-2 is an integral membrane Kunitz-type serine protease inhibitor that regulates the proteolysis of matriptase and prostasin in a cell-type selective manner. The cell-type selective nature of HAI-2 function depends largely on whether the inhibitor and potential target enzymes are targeted to locations in close proximity. The N-glycan moiety of HAI-2 can function as a subcellular targeting signal. HAI-2 is synthesized with 1 of 2 different N-glycan modifications: one of oligomannose-type, which largely remains in the endoplasmic reticulum/GA, and another of complex-type, which is targeted toward the apical surface in vesicle-like structures, and could function as an inhibitor of matriptase and prostasin. HAI-2 contains 2 putative N-glycosylation sites, Asn-57 and Asn-94, point mutations of which were generated and characterized in this study. The protein expression profile of the HAI-2 mutants indicates that Asn-57, and not Asn-94, is responsible for the N-glycosylation of both HAI-2 species, suggesting that the form with oligomannose-type N-glycan is the precursor of the form with complex-type N-glycan. Unexpectedly, the vast majority of non-glycosylated HAI-2 is synthesized into multiple disulfide-linked oligomers, which lack protease inhibitory function, likely due to distorted conformations caused by the disarrayed disulfide linkages. Although forced expression of HAI-2 in HAI-2 knockout cells artificially enhances HAI-2 oligomerization, disulfide-linked HAI-2 oligomers can also be observed in unmodified cells. These results suggest that N-glycosylation on Asn-57 is required for folding into a functional HAI-2 with full protease suppressive activity and correct subcellular targeting signal.

Single-chain multicolor-reporter templates for subcellular localization of molecular events in mammalian cells.

Single-chain multicolor-reporter imaging templates were developed for the subcellular localization of molecular events in mammalian cells. The templates were constructed by tandem linkage of fluorescent protein variants - fused with luciferases and the subcellular localization signal peptides. The templates simultaneously reported steroid hormonal activities at different optical spectra in the subcellular compartments. The templates contribute to the expansion of a toolbox of optical probes for subcellular localization of molecular events in intact cells.