Recognition and Chaperoning by Pex19, Followed by Trafficking and Membrane Insertion of the Peroxisome Proliferation Protein, Pex11

Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted therein. We demonstrate that Pex11 contains one Pex19-binding site (Pex19-BS) that is required for Pex11 insertion into peroxisomal membranes by Pex19, but is non-essential for peroxisomal trafficking. We provide extensive mutational analyses regarding the recognition of Pex19-BS in Pex11 by Pex19. Pex11 also has a second, Pex19-independent membrane peroxisome-targeting signal (mPTS) that is preserved among Pex11-family proteins and anchors the human HsPex11γ to the outer leaflet of the peroxisomal membrane. Thus, unlike most PMPs, Pex11 can use two mechanisms of transport to peroxisomes, where only one of them depends on its direct interaction with Pex19, but the other does not. However, Pex19 is necessary for membrane insertion of Pex11. We show that Pex11 can self-interact, using both homo- and/or heterotypic interactions involving its N-terminal helical domains. We demonstrate that Pex19 acts as a chaperone by interacting with the Pex19-BS in Pex11, thereby protecting Pex11 from spontaneous oligomerization that would otherwise cause its aggregation and subsequent degradation.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

The Viral Class II Membrane Fusion Machinery: Divergent Evolution from an Ancestral Heterodimer

A key step during the entry of enveloped viruses into cells is the merger of viral and cell lipid bilayers. This process is driven by a dedicated membrane fusion protein (MFP) present at the virion surface, which undergoes a membrane–fusogenic conformational change triggered by interactions with the target cell. Viral MFPs have been extensively studied structurally, and are divided into three classes depending on their three-dimensional fold. Because MFPs of the same class are found in otherwise unrelated viruses, their intra-class structural homology indicates horizontal gene exchange. We focus this review on the class II fusion machinery, which is composed of two glycoproteins that associate as heterodimers. They fold together in the ER of infected cells such that the MFP adopts a conformation primed to react to specific clues only upon contact with a target cell, avoiding premature fusion in the producer cell. We show that, despite having diverged in their 3D fold during evolution much more than the actual MFP, the class II accompanying proteins (AP) also derive from a distant common ancestor, displaying an invariant core formed by a β-ribbon and a C-terminal immunoglobulin-like domain playing different functional roles—heterotypic interactions with the MFP, and homotypic AP/AP contacts to form spikes, respectively. Our analysis shows that class II APs are easily identifiable with modern structural prediction algorithms, providing useful information in devising immunogens for vaccine design.

RNA length has a non-trivial effect in the stability of biomolecular condensates formed by RNA-binding proteins

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) play a crucial role in the spatiotemporal organization of the cell material. Nucleic acids can act as critical modulators in the stability of these protein condensates. Here, we present a multiscale computational strategy, exploiting the advantages of both a sequence-dependent coarse-grained representation of proteins and a minimal coarse-grained model that represent proteins as patchy colloids, to unveil the role of RNA length in regulating the stability of RNA-binding protein (RBP) condensates. We find that for a constant RNA/protein ratio in which phase separation is enhanced, the protein fused in sarcoma (FUS), which can phase separate on its own-i.e., via homotypic interactions-only exhibits a mild dependency on the RNA strand length, whereas, the 25-repeat proline-arginine peptide (PR25), which does not undergo LLPS on its own at physiological conditions but instead exhibits complex coavervation with RNA-i.e., via heterotypic interactions-shows a strong dependence on the length of the added RNA chains. Our minimal patchy particle simulations, where we recapitulate the modulation of homotypic protein LLPS and complex coacervation by RNA length, suggest that the strikingly different effect of RNA length on homotypic LLPS versus complex coacervation is general. Phase separation is RNA-length dependent as long as the relative contribution of heterotypic interactions sustaining LLPS is comparable or higher than that committed by protein homotypic interactions. Taken together, our results contribute to illuminate the intricate physicochemical mechanisms that influence the stability of RBP condensates through RNA inclusion.

Spatiotemporal Modulations in Heterotypic Condensates of Prion and α-Synuclein Control Phase Transitions and Amyloid Conversion

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) of proteins and nucleic acids are thought to govern critical cellular functions. These multicomponent assemblies provide dynamic hubs for competitive homotypic and heterotypic interactions. Here, we demonstrate that the complex coacervation between the prion protein (PrP) and α-synuclein (α-Syn) within a narrow stoichiometry regime results in the formation of highly dynamic liquid droplets. Domain-specific electrostatic interactions between the positively charged intrinsically disordered N-terminal segment of PrP and the negatively charged C-terminal domain of α-Syn drive the formation of these highly tunable, reversible, thermo-responsive condensates. Picosecond time-resolved measurements revealed the existence of relatively ordered electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making on a much slower timescale giving rise to the liquid-like behavior on the second timescale and mesoscopic length-scale. The addition of RNA to these preformed coacervates yields multiphasic, anisotropic, vesicle-like, hollow condensates. LLPS promotes liquid-to-solid maturation of α-Syn-PrP condensates resulting in the rapid conversion into heterotypic amyloids. Our results suggest that synergistic interactions between PrP and α-Syn in liquid condensates can offer mechanistic underpinnings of their physiological role and overlapping neuropathological features.

O11 Activated leukocyte cell adhesion molecule (ALCAM/CD166) is an important clinical and prognostic indicator in pancreatic cancer

Introduction Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a cell adhesion molecule and one of the potential metastasis ‘soil’ receptors that via homotypic and heterotypic interactions, mediates cell adhesion. The present study investigated the clinical, pathological and prognostic value of ALCAM in patients with pancreatic cancer. Method Pancreatic cancer tissues (n = 223), collected immediately after surgery, were analysed for levels of the ALCAM transcripts. The expression was analysed against clinical, pathological and clinical outcome of the patients. We validated our findings with an available TCGA database (n = 117), including correlations with the ALCAM interactive partners. Result Pancreatic cancer tissues had significantly higher levels of ALCAM transcript than normal tissues (P < 0.00001). There were no significant differences with staging, differentiation and tumour locations. Tumours from patients who died of pancreatic cancer had significantly high levels of ALCAM compared with those who lived (P = 0.018), finding also supported by ROC analysis (P = 0.016). Multivariant analysis showed ALCAM as an independent prognosis factor for overall survival (hazardous ratio 5.485), with both nodal status and TNM staging contributing to the model (HR 2.578 and 3.02 respectively). A surprising finding was the relationship between ALCAM expression and microvessel embolism of tumour cells (P = 0.021, with vs without tumour embolism). ALCAM significantly correlated with its interactive protein partners including CD6 and ITGB1, but not L1CAM. Conclusion ALCAM/CD166 expression is aberrant in pancreatic cancer and the raised expression is an independent prognostic factor for the survival of the patients and the microvessel embolism by cancer cells. Take-home Message ALCAM is a prognostic indicator for survival and tumour embolism in pancreatic cancer

Physical theory of biological noise buffering by multicomponent phase separation

Maintaining homeostasis is a fundamental characteristic of living systems. In cells, this is contributed to by the assembly of biochemically distinct organelles, many of which are not membrane bound but form by the physical process of liquid–liquid phase separation (LLPS). By analogy with LLPS in binary solutions, cellular LLPS was hypothesized to contribute to homeostasis by facilitating “concentration buffering,” which renders the local protein concentration within the organelle robust to global variations in the average cellular concentration (e.g., due to expression noise). Interestingly, concentration buffering was experimentally measured in vivo in a simple organelle with a single solute, while it was observed not to be obeyed in one with several solutes. Here, we formulate theoretically and solve analytically a physical model of LLPS in a ternary solution of two solutes (ϕ and ψ) that interact both homotypically (ϕ–ϕ attractions) and heterotypically (ϕ–ψ attractions). Our physical theory predicts how the coexisting concentrations in LLPS are related to expression noise and thus, generalizes the concept of concentration buffering to multicomponent systems. This allows us to reconcile the seemingly contradictory experimental observations. Furthermore, we predict that incremental changes of the homotypic and heterotypic interactions among the molecules that undergo LLPS, such as those that are caused by mutations in the genes encoding the proteins, may increase the efficiency of concentration buffering of a given system. Thus, we hypothesize that evolution may optimize concentration buffering as an efficient mechanism to maintain LLPS homeostasis and suggest experimental approaches to test this in different systems.

Neuroinflammation: Integrated Nervous Tissue Response through Intercellular Interactions at the “Whole System” Scale

Different cell populations in the nervous tissue establish numerous, heterotypic interactions and perform specific, frequently intersecting activities devoted to the maintenance of homeostasis. Microglia and astrocytes, respectively the immune and the “housekeeper” cells of nervous tissue, play a key role in neurodegenerative diseases. Alterations of tissue homeostasis trigger neuroinflammation, a collective dynamic response of glial cells. Reactive astrocytes and microglia express various functional phenotypes, ranging from anti-inflammatory to pro-inflammatory. Chronic neuroinflammation is characterized by a gradual shift of astroglial and microglial phenotypes from anti-inflammatory to pro-inflammatory, switching their activities from cytoprotective to cytotoxic. In this scenario, the different cell populations reciprocally modulate their phenotypes through intense, reverberating signaling. Current evidence suggests that heterotypic interactions are links in an intricate network of mutual influences and interdependencies connecting all cell types in the nervous system. In this view, activation, modulation, as well as outcomes of neuroinflammation, should be ascribed to the nervous tissue as a whole. While the need remains of identifying further links in this network, a step back to rethink our view of neuroinflammation in the light of the “whole system” scale, could help us to understand some of its most controversial and puzzling features.

The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

Heterotypic interactions between newly transformed cells and normal surrounding cells define tumor’s fate in incipient carcinomas. Once homeostasis has been lost, normal resident fibroblasts become carcinoma-associated fibroblasts, conferring protumorogenic properties on these normal cells. Here we describe the IL1β-mediated interplay between cancer cells and normal colonic myofibroblasts (NCFs), which bestows differential sensitivity to cytotoxic drugs on tumor cells. We used NCFs, their conditioned media (CM), and cocultures with tumor cells to characterize the IL1β-mediated crosstalk between both cell types. We silenced IL1β in tumor cells to demonstrate that such cells do not exert an influence on NCFs inflammatory phenotype. Our results shows that IL1β is overexpressed in cocultured tumor cells. IL1β enables paracrine signaling in myofibroblasts, converting them into inflammatory-CAFs (iCAF). IL1β-stimulated-NCF-CM induces migration and differential sensitivity to oxaliplatin in colorectal tumor cells. Such chemoprotective effect has not been evidenced for TGFβ1-driven NCFs. IL1β induces the loss of a myofibroblastic phenotype in NCFs and acquisition of iCAF traits. In conclusion, IL1β-secreted by cancer cells modify surrounding normal fibroblasts to confer protumorogenic features on them, particularly tolerance to cytotoxic drugs. The use of IL1β-blocking agents might help to avoid the iCAF traits acquisition and consequently to counteract the protumorogenic actions these cells.