Methylmalonic acidemia triggers lysosomal-autophagy dysfunctions.

BACKGROUND: Methylmalonic acidemia (MMA) is a rare inborn error of propionate metabolism caused by deficiency of the mitochondrial methylmalonyl-CoA mutase (MUT) enzyme. As matter of fact, MMA patients manifest impairment of the primary metabolic network with profound damages that involve several cell components, many of which have not been discovered yet. We employed cellular models and patients-derived fibroblasts to refine and uncover new pathologic mechanisms connected with MUT deficiency through the combination of multi-proteomics and bioinformatics approaches. RESULTS: Our data show that MUT deficiency is connected with profound proteome dysregulations, revealing molecular actors involved in lysosome and autophagy functioning. To elucidate the effects of defective MUT on lysosomal and autophagy regulation, we analyzed the morphology and functionality of MMA-lysosomes that showed deep alterations, thus corroborating omics data. Lysosomes of MMA cells present as enlarged vacuoles with low degradative capabilities. Notwithstanding, treatment with an anti-propionigenic drug is capable of totally rescuing lysosomal morphology and functional activity in MUT-deficient cells. These results indicate a strict connection between MUT deficiency and lysosomal-autophagy dysfunction, providing promising therapeutic perspectives for MMA. CONCLUSIONS: Defective homeostatic mechanisms in the regulation of autophagy and lysosome functions have been demonstrated in MUT-deficient cells. Our data prove that MMA triggers such dysfunctions impacting on autophagosome-lysosome fusion and lysosomal activity.

Metabolic rewiring and autophagy inhibition correct lysosomal storage disease in mucopolysaccharidosis IIIB.

Mucopolysaccharidoses (MPSs) are lysosomal disorders with neurological involvement for which no cure exists. Here, we show that recombinant NK1 fragment of hepatocyte growth factor rescues substrate accumulation and lysosomal defects in MPS I, IIIA and IIIB patient fibroblasts. We investigated PI3K/Akt pathway, which is of crucial importance for neuronal function and survival, and demonstrate that PI3K inhibition abolishes NK1 therapeutic effects. We identified that autophagy inhibition, by Beclin1 silencing, reduces MPS IIIB phenotype and that NK1 downregulates autophagic-lysosome (ALP) gene expression, suggesting a possible contribution of autophagosome biogenesis in MPS. Indeed, metabolomic analyses revealed defects of mitochondrial activity accompanied by anaerobic metabolism and inhibition of AMP-activated protein kinase (AMPK), which acts on metabolism and autophagy, rescues lysosomal defects. These results provide insights into the molecular mechanisms of MPS IIIB physiopathology, supporting the development of new promising approaches based on autophagy inhibition and metabolic rewiring to correct lysosomal pathology in MPSs.

Label-Free Intracellular Multi-Specificity in Yeast Cells by Phase-Contrast Tomographic Flow Cytometry.

In-flow phase-contrast tomography provides a 3D refractive index of label-free cells in cytometry systems. Its major limitation, as with any quantitative phase imaging approach, is the lack of specificity compared to fluorescence microscopy, thus restraining its huge potentialities in single-cell analysis and diagnostics. Remarkable results in introducing specificity are obtained through artificial intelligence (AI), but only for adherent cells. However, accessing the 3D fluorescence ground truth and obtaining accurate voxel-level co-registration of image pairs for AI training is not viable for high-throughput cytometry. The recent statistical inference approach is a significant step forward for label-free specificity but remains limited to cells' nuclei. Here, a generalized computational strategy based on a self-consistent statistical inference to achieve intracellular multi-specificity is shown. Various subcellular compartments (i.e., nuclei, cytoplasmic vacuoles, the peri-vacuolar membrane area, cytoplasm, vacuole-nucleus contact site) can be identified and characterized quantitatively at different phases of the cells life cycle by using yeast cells as a biological model. Moreover, for the first time, virtual reality is introduced for handling the information content of multi-specificity in single cells. Full fruition is proofed for exploring and interacting with 3D quantitative biophysical parameters of the identified compartments on demand, thus opening the route to a metaverse for 3D microscopy.

Scaffold proteins of cancer signaling networks: The paradigm of FK506 binding protein 51 (FKBP51) supporting tumor intrinsic properties and immune escape.

Scaffold proteins are crucial regulators of signaling networks, and their abnormal expression may favor the development of tumors. Among the scaffold proteins, immunophilin covers a unique role as 'protein-philin' (Greek 'philin' = friend) that interacts with proteins to guide their proper assembly. The growing list of human syndromes associated with the immunophilin defect underscores the biological relevance of these proteins that are largely opportunistically exploited by cancer cells to support and enable the tumor's intrinsic properties. Among the members of the immunophilin family, the FKBP5 gene was the only one identified to have a splicing variant. Cancer cells impose unique demands on the splicing machinery, thus acquiring a particular susceptibility to splicing inhibitors. This review article aims to overview the current knowledge of the FKBP5 gene functions in human cancer, illustrating how cancer cells exploit the scaffolding function of canonical FKBP51 to foster signaling networks that support their intrinsic tumor properties and the spliced FKBP51s to gain the capacity to evade the immune system.

Alternative splicing of FKBP5 gene exerts control over T lymphocyte expansion.

FKBP51 is constitutively expressed by immune cells. As other FKBP family members, FKBP51 acts as a coreceptor for the natural products FK506 and rapamycin, which exhibit immunosuppressive effects. However, little is known about the intrinsic role of this large FKBP in the primary function of lymphocytes, that is, the adaptive immune response against foreign antigens, for example, pathogens. This paper aimed to investigate whether FKBP51 expression was modulated during lymphocyte activation. Moreover, as we recently identified a splicing isoform of FKBP51, namely FKBP51s, we also measured this splice protein, along with the canonical one, at different times of a peripheral blood mononuclear cell culture stimulated via T cell receptor. Our results show that the two FKBP51 isoforms were alternatively induced during the proliferative burst. Canonical FKBP51 increased in the time window between 48 and 96 h and its expression levels correlated with cyclin D levels. FKBP51s transiently increased earlier, at 24-36 h to reappearing later, at 120 h, when cyclin D expression returned at resting levels and proliferation ceased. Interestingly, within these two specific timeframes, FKBP51s accumulated in the nucleus. Here FKBP51s colocalized with the Foxp3 transcription factor at 36 h. Regulatory T cell (Treg) counts significantly decreased when FKBP51s was downmodulated. The coculture suppression assay suggested that FKBP51s supports the suppressive capability of Tregs. At 120 h, chromatin immunoprecipitation experiments found FKBP51s linked to CCND1 gene, suggesting a possible effect on gene transcription regulation, as previously demonstrated in melanoma. In conclusion, our study shows that FKBP5 isoforms are upregulated during lymphocyte activation, albeit on different timeframes. The activation of canonical FKBP51 coincides with proliferation hallmarks; FKBP5 splicing occurs early to sustain Treg development and late when proliferation ceases.

N-Substituted l-Iminosugars for the Treatment of Sanfilippo Type B Syndrome.

Sanfilippo syndrome comprises a group of four genetic diseases due to the lack or decreased activity of enzymes involved in heparan sulfate (HS) catabolism. HS accumulation in lysosomes and other cellular compartments results in tissue and organ dysfunctions, leading to a wide range of clinical symptoms including severe neurodegeneration. To date, no approved treatments for Sanfilippo disease exist. Here, we report the ability of N-substituted l-iminosugars to significantly reduce substrate storage and lysosomal dysfunctions in Sanfilippo fibroblasts and in a neuronal cellular model of Sanfilippo B subtype. Particularly, we found that they increase the levels of defective α-N-acetylglucosaminidase and correct its proper sorting toward the lysosomal compartment. Furthermore, l-iminosugars reduce HS accumulation by downregulating protein levels of exostosin glycosyltransferases. These results highlight an interesting pharmacological potential of these glycomimetics in Sanfilippo syndrome, paving the way for the development of novel therapeutic approaches for the treatment of such incurable disease.

The Lack of STING Impairs the MHC-I Dependent Antigen Presentation and JAK/STAT Signaling in Murine Macrophages.

STING is a transmembrane ER resident protein that was initially described as a regulator of innate immune response triggered by viral DNA and later found to be involved in a broader range of immune processes. Here, we assessed its role in the antigen presentation by generating a STING KO macrophage cell line. In the absence of STING, we observed an impaired OVA-derived SIINFEKL peptide presentation together with a decreased level of MHC-I complex on the plasma membrane, likely due to a decreased mRNA expression of ß2 m light chain as no relevant alterations of the peptide-loading complex (TAPs) were found. Moreover, JAK-STAT signaling resulted in impaired STING KO cells following OVA and LPS treatments, suggesting a dampened activation of immune response. Our data revealed a new molecular role of STING in immune mechanisms that could elucidate its role in the pathogenesis of autoimmune disorders and cancer.

Lysosomal positioning diseases: beyond substrate storage.

Lysosomal storage diseases (LSDs) comprise a group of inherited monogenic disorders characterized by lysosomal dysfunctions due to undegraded substrate accumulation. They are caused by a deficiency in specific lysosomal hydrolases involved in cellular catabolism, or non-enzymatic proteins essential for normal lysosomal functions. In LSDs, the lack of degradation of the accumulated substrate and its lysosomal storage impairs lysosome functions resulting in the perturbation of cellular homeostasis and, in turn, the damage of multiple organ systems. A substantial number of studies on the pathogenesis of LSDs has highlighted how the accumulation of lysosomal substrates is only the first event of a cascade of processes including the accumulation of secondary metabolites and the impairment of cellular trafficking, cell signalling, autophagic flux, mitochondria functionality and calcium homeostasis, that significantly contribute to the onset and progression of these diseases. Emerging studies on lysosomal biology have described the fundamental roles of these organelles in a variety of physiological functions and pathological conditions beyond their canonical activity in cellular waste clearance. Here, we discuss recent advances in the knowledge of cellular and molecular mechanisms linking lysosomal positioning and trafficking to LSDs.

Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells.

The lysosomal compartment is a key hub for cell metabolism, and morphological alterations have been described in several pathological conditions. Here, we describe the use of amino acid analogs modified by the presence of a methyl ester group that accumulates within lysosomes. This generates an intraluminal osmotic effect able to trigger a rapid and selective expansion of the lysosomal compartment within 2 h of treatment. We also present protocols to preserve lysosomal morphology, which yields a more accurate size measurement. For complete details on the use and execution of this protocol, please refer to Scerra et al. (2021).

Metabolites Profiling of Melanoma Interstitial Fluids Reveals Uridine Diphosphate as Potent Immune Modulator Capable of Limiting Tumor Growth.

Tumor interstitial fluid (TIF) surrounds and perfuses tumors and collects ions, metabolites, proteins, and extracellular vesicles secreted by tumor and stromal cells. Specific metabolites, accumulated within the TIF, could induce metabolic alterations of immune cells and shape the tumor microenvironment. We deployed a metabolomic approach to analyze the composition of melanoma TIF and compared it to the plasma of C57BL6 mice, engrafted or not with B16-melanoma cells. Among the classes of metabolites analyzed, monophosphate and diphosphate nucleotides resulted enriched in TIF compared to plasma samples. The analysis of the effects exerted by guanosine diphosphate (GDP) and uridine diphosphate (UDP) on immune response revealed that GDP and UDP increased the percentage of CD4+CD25+FoxP3- and, on isolated CD4+ T-cells, induced the phosphorylation of ERK, STAT1, and STAT3; increased the activity of NF-κB subunits p65, p50, RelB, and p52; increased the expression of Th1/Th17 markers including IFNγ, IL17, T-bet, and RORγt; and reduced the expression of IL13, a Th2 marker. Finally, we observed that local administrations of UDP in B16-engrafted C57BL6 mice reduced tumor growth and necrotic areas. In addition, UDP-treated tumors showed a higher presence of MHCIIhi tumor-associated macrophage (TAM) and of CD3+CD8+ and CD3+CD4+ tumor-infiltrating T-lymphocytes (TILs), both markers of anti-tumor immune response. Consistent with this, intra-tumoral gene expression analysis revealed in UDP-treated tumors an increase in the expression of genes functionally linked to anti-tumor immune response. Our analysis revealed an important metabolite acting as mediator of immune response, which could potentially represent an additional tool to be used as an adjuvant in cancer immunotherapy.

PD-L1 Expression Fluctuates Concurrently with Cyclin D in Glioblastoma Cells.

Despite Glioblastoma (GBM) frequently expressing programmed cell death ligand-1 (PD-L1), treatment with anti-programmed cell death-1 (PD1) has not yielded brilliant results. Intratumor variability of PD-L1 can impact determination accuracy. A previous study on mouse embryonic fibroblasts (MEFs) reported a role for cyclin-D in control of PD-L1 expression. Because tumor-cell growth within a cancer is highly heterogeneous, we looked at whether PD-L1 and its cochaperone FKBP51s were influenced by cell proliferation, using U251 and SF767 GBM-cell-lines. PD-L1 was measured by Western blot, flow cytometry, confocal-microscopy, quantitative PCR (qPCR), CCND1 by qPCR, FKBP51s by Western blot and confocal-microscopy. Chromatin-Immunoprecipitation assay (xChIp) served to assess the DNA-binding of FKBP51 isoforms. In the course of cell culture, PD-L1 appeared to increase concomitantly to cyclin-D on G1/S transition, to decrease during exponential cell growth progressively. We calculated a correlation between CCND1 and PD-L1 gene expression levels. In the temporal window of PD-L1 and CCND1 peak, FKBP51s localized in ER. When cyclin-D declined, FKBP51s went nuclear. XChIp showed that FKBP51s binds CCND1 gene in a closed-chromatin configuration. Our finding suggests that the dynamism of PD-L1 expression in GBM follows cyclin-D fluctuation and raises the hypothesis that FKBP51s might participate in the events that govern cyclin-D oscillation.

Early onset effects of single substrate accumulation recapitulate major features of LSD in patient-derived lysosomes.

Lysosome functions mainly rely on their ability to either degrade substrates or release them into the extracellular space. Lysosomal storage disorders (LSDs) are commonly characterized by a chronic lysosomal accumulation of different substrates, thereby causing lysosomal dysfunctions and secretion defects. However, the early effects of substrate accumulation on lysosomal homeostasis have not been analyzed so far. Here, we describe how the acute accumulation of a single substrate determines a rapid centripetal redistribution of the lysosomes, triggering their expansion and reducing their secretion, by limiting the motility of these organelles toward the plasma membrane. Moreover, we provide evidence that such defects could be explained by a trapping mechanism exerted by the extensive contacts between the enlarged lysosomes and the highly intertwined membrane structures of the endoplasmic reticulum which might represent a crucial biological cue ultimately leading to the clinically relevant secondary defects observed in the LSD experimental models and patients.

Competitive binding of extracellular accumulated heparan sulfate reduces lysosomal storage defects and triggers neuronal differentiation in a model of Mucopolysaccharidosis IIIB.

Mucopolysaccharidoses (MPSs) are a group of inherited lysosomal storage disorders associated with the deficiency of lysosomal enzymes involved in glycosaminoglycan (GAG) degradation. The resulting cellular accumulation of GAGs is responsible for widespread tissue and organ dysfunctions. The MPS III, caused by mutations in the genes responsible for the degradation of heparan sulfate (HS), includes four subtypes (A, B, C, and D) that present significant neurological manifestations such as progressive cognitive decline and behavioral disorders. The established treatments for the MPS III do not cure the disease but only ameliorate non-neurological clinical symptoms. We previously demonstrated that the natural variant of the hepatocyte growth factor NK1 reduces the lysosomal pathology and reactivates impaired growth factor signaling in fibroblasts from MPS IIIB patients. Here, we show that the recombinant NK1 is effective in rescuing the morphological and functional dysfunctions of lysosomes in a neuronal cellular model of the MPS IIIB. More importantly, NK1 treatment is able to stimulate neuronal differentiation of neuroblastoma SK-NBE cells stable silenced for the NAGLU gene causative of the MPS IIIB. These results provide the basis for the development of a novel approach to possibly correct the neurological phenotypes of the MPS IIIB as well as of other MPSs characterized by the accumulation of HS and progressive neurodegeneration.

Fusion Pores Live on the Edge.

Biological transmission of vesicular content occurs by opening of a fusion pore. Recent experimental observations have illustrated that fusion pores between vesicles that are docked by an extended flat contact zone are located at the edge (vertex) of this zone. We modeled this experimentally observed scenario by coarse-grained molecular simulations and elastic theory. This revealed that fusion pores experience a direct attraction toward the vertex. The size adopted by the resulting vertex pore strongly depends on the apparent contact angle between the adhered vesicles even in the absence of membrane surface tension. Larger contact angles substantially increase the equilibrium size of the vertex pore. Because the cellular membrane fusion machinery actively docks membranes, it facilitates a collective expansion of the contact zone and increases the contact angle. In this way, the fusion machinery can drive expansion of the fusion pore by free energy equivalents of multiple tens of kBT from a distance and not only through the fusion proteins that reside within the fusion pore.

Effects of Long-Term Citrate Treatment in the PC3 Prostate Cancer Cell Line.

Acute administration of a high level of extracellular citrate displays an anti-proliferative effect on both in vitro and in vivo models. However, the long-term effect of citrate treatment has not been investigated yet. Here, we address this question in PC3 cells, a prostate-cancer-derived cell line. Acute administration of high levels of extracellular citrate impaired cell adhesion and inhibited the proliferation of PC3 cells, but surviving cells adapted to grow in the chronic presence of 20 mM citrate. Citrate-resistant PC3 cells are significantly less glycolytic than control cells. Moreover, they overexpress short-form, citrate-insensitive phosphofructokinase 1 (PFK1) together with full-length PFK1. In addition, they show traits of mesenchymal-epithelial transition: an increase in E-cadherin and a decrease in vimentin. In comparison with PC3 cells, citrate-resistant cells display morphological changes that involve both microtubule and microfilament organization. This was accompanied by changes in homeostasis and the organization of intracellular organelles. Thus, the mitochondrial network appears fragmented, the Golgi complex is scattered, and the lysosomal compartment is enlarged. Interestingly, citrate-resistant cells produce less total ROS but accumulate more mitochondrial ROS than control cells. Consistently, in citrate-resistant cells, the autophagic pathway is upregulated, possibly sustaining their survival. In conclusion, chronic administration of citrate might select resistant cells, which could jeopardize the benefits of citrate anticancer treatment.

Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles.

Fluorescence de-quenching can be used to analyze membrane lipid mixing during an in vitro fusion reaction. Here we describe a method to measure lipid mixing using vacuolar membranes purified from the yeast Saccharomyces cerevisiae. Labeling the isolated organelles with rhodamine-phosphatidylethanolamine allows to reveal ATP-dependent lipid mixing through fluorescence de-quenching in a spectrofluorometer. Combining this assay with content mixing indicators, such as the fusion-dependent maturation of a luminal vacuolar phosphatase, then permits the detection of hemifusion intermediates and the analysis of the requirements for fusion pore opening.

SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle.

Constitutive membrane fusion within eukaryotic cells is thought to be controlled at its initial steps, membrane tethering and SNARE complex assembly, and to rapidly proceed from there to full fusion. Although theory predicts that fusion pore expansion faces a major energy barrier and might hence be a rate-limiting and regulated step, corresponding states with non-expanding pores are difficult to assay and have remained elusive. Here, we show that vacuoles in living yeast are connected by a metastable, non-expanding, nanoscopic fusion pore. This is their default state, from which full fusion is regulated. Molecular dynamics simulations suggest that SNAREs and the SM protein-containing HOPS complex stabilize this pore against re-closure. Expansion of the nanoscopic pore to full fusion can thus be triggered by osmotic pressure gradients, providing a simple mechanism to rapidly adapt organelle volume to increases in its content. Metastable, nanoscopic fusion pores are then not only a transient intermediate but can be a long-lived, physiologically relevant and regulated state of SNARE-dependent membrane fusion.

An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population.

The H1069Q substitution is the most frequent mutation of the Cu transporter ATP7B that causes Wilson disease in the Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes, but, in the presence of excessive Cu, it relocates to the endo-lysosomal compartment to excrete Cu via bile canaliculi. In contrast, ATP7B-H1069Q is strongly retained in the ER, does not reach the Golgi complex and fails to move to the endo-lysosomal compartment in the presence of excessive Cu, thus causing toxic Cu accumulation. We have previously shown that, in transfected cells, the small heat-shock protein αB-crystallin is able to correct the mislocalization of ATP7B-H1069Q and its trafficking in the presence of Cu overload. Here, we first show that the α-crystallin domain of αB-crystallin mimics the effect of the full-length protein, whereas the N- and C-terminal domains have no such effect. Next, and most importantly, we demonstrate that a twenty-residue peptide derived from the α-crystallin domain of αB-crystallin fully rescues Golgi localization and the trafficking response of ATP7B-H1069Q in the presence of Cu overload. In addition, we show that this peptide interacts with the mutant transporter in the live cell. These results open the way to attempt developing a pharmacologically active peptide to specifically contrast the Wilson disease form caused by the ATP7B-H1069Q mutant.

A tethering complex drives the terminal stage of SNARE-dependent membrane fusion.

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signalling molecules, such as hormones and neurotransmitters. Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Tethering proteins mediate the initial recognition and attachment of membranes, whereas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes are considered as the core fusion engine. SNARE complexes provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore. This last step is highly energy-demanding. Here we combine the in vivo and in vitro fusion of yeast vacuoles with molecular simulations to show that tethering proteins are critical for overcoming the final energy barrier to fusion pore formation. SNAREs alone drive vacuoles only into the hemifused state. Tethering proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening and provides the driving force. Thereby, tethering proteins assume a crucial mechanical role in the terminal stage of membrane fusion that is likely to be conserved at multiple steps of vesicular traffic. We therefore propose that SNAREs and tethering proteins should be considered as a single, non-dissociable device that drives fusion. The core fusion machinery may then be larger and more complex than previously thought.

Differential phosphorylation-based regulation of αB-crystallin chaperone activity for multipass transmembrane proteins.

We have previously shown that αB-crystallin (CRYAB), a small heat shock protein (sHsp) that prevents irreversible aggregation of unfolded protein by an ATP-independent chaperone activity, plays a pivotal role in the biogenesis of multipass transmembrane proteins (TMPs) assisting their folding from the cytosolic side of the endoplasmic reticulum (ER) (D'Agostino et al., 2013). Here we present evidence, based on phosphomimetic substitutions, that the three phosphorytable serine residues at position 19, 45 and 59 of CRYAB play a different regulatory role in this novel chaperone activity: S19 and S45 have a strong inhibitory effect, either alone or in combination, while S59 has not and counteracts the inhibition caused by single phosphomimetic substitutions at S19 and S45. Interestingly, all phosphomimetic substitutions determine the formation of smaller oligomeric complexes containing CRYAB, indicating that the inhibitory effect seen for S19 and S45 cannot be ascribed to the reduction of oligomerization frequently associated to a decreased chaperone activity. These results indicate that phosphorylation finely regulates the chaperone activity of CRYAB with multipass TMPs and suggest a pivotal role for S59 in this process.