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The kidney-specific gene

The kidney-specific gene UMOD encodes for uromodulin, the most abundant protein excreted in normal urine. Rare, large-effect variants in UMOD cause autosomal dominant tubulointerstitial kidney disease (ADTKD) while common, low-effect variants strongly associate with kidney function and risk of chronic kidney disease (CKD) in the general population. It is unknown whether intermediate-effect variants in UMOD contribute to CKD. Here, candidate intermediate-effect UMOD variants were identified using large population and ADTKD cohorts. Biological and phenotypical effects were investigated using cell models, in silico simulations and international databases and biobanks. Eight UMOD missense variants reported in ADTKD are present in gnomAD with MAF ranging from 10−5 to 10−3. Among them, the missense variant p.Thr62Pro is detected in ∼1/1,000 individuals of European ancestry, shows incomplete penetrance but a high genetic load in familial clusters of CKD and is associated with kidney failure in the 100,000 Genomes Project (OR 3.99; 1.84-8.98) and the UK Biobank (OR 4.12; 1.32-12.85). Compared to canonical ADTKD mutations, the p.Thr62Pro carriers displayed reduced disease severity, with slower progression of CKD, intermediate reduction of urinary UMOD levels, in line with an intermediate trafficking defect in vitro. Identification of an intermediate-effect UMOD variant completes the spectrum of UMOD-associated kidney diseases and provides novel insights into the mechanisms of ADTKD and the genetic architecture of CKD.Significance StatementThe genetic architecture of chronic kidney disease (CKD) remains incompletely understood. Variants in the kidney-specific gene UMOD cause autosomal dominant tubulointerstitial kidney disease (ADTKD) and are associated with kidney function and risk of CKD in the general population. Here, we identified an intermediate-effect variant, p.Thr62Pro, detected in ∼1:1,000 individuals of European ancestry, that showed a high genetic load in familial clusters of CKD and was associated with an OR of ∼4 for kidney failure in the 100,000 Genomes Project and the UK Biobank. Compared to canonical ADTKD mutations, p.Thr62Pro carriers displayed reduced disease severity and an intermediate trafficking defect. These findings complete the spectrum of UMOD-associated kidney diseases and provide a paradigm for the genetic contribution to CKD.

FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer. Fucci et al. show that the poly(A) polymerase FAM46C acts as a multiple myeloma-specific tumor suppressor, increasing secretory capacity and antibody production beyond sustainability via its interaction with endoplasmic reticulum transmembrane FNDC3 proteins. Moreover, its activity is restricted through proteasomal degradation or p62-dependent aggregation and sequestration from FNDC3 proteins.

ADAMo (Adaptive Digital Aerodynamics for Motorcycles) is a research project guided by Piaggio S.p.A and partially founded by Toscana region. The object was to increase the performance, safety and comfort of a motorcycle by using active aerodynamics devices.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a genetically heterogeneous renal disorder leading to progressive loss of renal function. ADTKD-REN is due to rare mutations in renin, all localized in the protein leader peptide and affecting its co-translational insertion in the endoplasmic reticulum (ER). Through exome sequencing in an adult-onset ADTKD family we identified a new renin variant, p.L381P, mapping in the mature protein. To assess its pathogenicity, we combined genetic data, computational and predictive analysis and functional studies. The L381P substitution affects an evolutionary conserved residue, co-segregates with renal disease, is not found in population databases and is predicted to be deleterious by in silico tools and by structural modelling. Expression of the L381P variant leads to its ER retention and induction of the Unfolded Protein Response in cell models and to defective pronephros development in zebrafish. Our work shows that REN mutations outside of renin leader peptide can cause ADTKD and delineates an adult form of ADTKD-REN, a condition which has usually its onset in childhood. This has implications for the molecular diagnosis and the estimated prevalence of the disease and points at ER homeostasis as a common pathway affected in ADTKD-REN, and possibly more generally in ADTKD.

Proteasome inhibitors (PIs) are effective against multiple myeloma (MM), but the mechanisms of action and bases of individual susceptibility remain unclear. Recent work linked PI sensitivity to protein synthesis and proteasome activity, raising the question whether different levels of proteasome expression and workload underlie PI sensitivity in MM cells (MMCs). Exploiting human MM lines characterized by differential PI sensitivity, we report that highly sensitive MMCs express lower proteasome levels and higher proteasomal workload than relatively PI-resistant MMCs, resulting in the accumulation of polyubiquitinated proteins at the expense of free ubiquitin (proteasome stress). Manipulating proteasome expression or workload alters apoptotic sensitivity to PI, demonstrating a cause-effect relationship between proteasome stress and apoptotic responses in MMCs. Intracellular immunostaining in primary, patient-derived MMCs reveals that polyubiquitinated proteins hallmark neoplastic plasma cells, in positive correlation with immunoglobulin (Ig) content, both intra- and interpatient. Moreover, overall proteasome activity of primary MMCs inversely correlates with apoptotic sensitivity to PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMCs to PI, potentially providing a framework for identifying indicators of responsiveness and designing novel combination therapies.

Caveolin-1 and caveolin-2 are related proteins involved in the biogenesis of caveolae. The corresponding genes in humans (CAV and CAV2, respectively), have been mapped to a common locus in chromosome 7q31.1, and are possible candidates for the tumor suppressor gene postulated in this region. Here, we show that CAV and CAV2 are independent transcriptional units lying in the same orientation, with CAV2 centromeric and about 17 kb upstream to CAV. The two genes have similar tissue expression patterns. Alternative termination/polyadenylation generates two CAV2 mRNAs. Multiple transcriptional start sites spanning 35 bp upstream from the CAV2 ATG are detected by 5′ RACE, consistent with a TATA-less promoter predicted by sequence analysis. The CAV2 promoter region contains two SRE-like boxes resembling those described in the CAV promoter and proposed to link transcription to intracellular cholesterol levels. However, exogenous sterols had only minor effects on CAV and CAV2 RNA levels in HeLa cells, suggesting that SREBPs are not sufficient to regulate caveolin transcription.

The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5(-/-) differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.

Protein synthesis determines sensitivity of differentiating and malignant plasma cells to proteasome inhibitors by saturating cellular proteolytic capacity. A previously unsuspected, considerable proportion of newly synthesized polypeptides are hydrolyzed rapidly by proteasomes, possibly competing with endogenous substrates and altering proteostasis. In view of the anti-cancer effects of PIs, we set out to achieve a quantitative assessment of proteasome workload in cells hallmarked by different PI sensitivity, namely, a panel of MM cells, and in a dynamic model of plasma cell differentiation, a process that confers exquisite PI sensitivity. Our results suggest that protein synthesis is a key determinant of proteasomal proteolytic burden and PI sensitivity. In different MM cells and in differentiating plasma cells, the average proteolytic work accomplished per proteasome ranges over different orders of magnitude, an unexpected degree of variability, with increased workload invariably associated to increased PI sensitivity. The unfavorable load-versus-capacity balance found in highly PI-sensitive MM lines is accounted for by a decreased total number of immunoproteasomes/cell coupled to enhanced generation of RDPs. Moreover, indicative of cause-effect relationships, attenuating general protein synthesis by the otherwise toxic agent CHX reduces PI sensitivity in activated B and in MM cells. Our data support the view that in plasma cells protein synthesis contributes to determine PI sensitivity by saturating the proteasomal degradative capacity. Quantitating protein synthesis and proteasome workload may thus prove crucial to design novel negative proteostasis regulators against cancer.

Proteasome inhibitors (PI) proved to be extremely effective against different types of cancer, particularly against Multiple Myeloma (MM), a frequent and still incurable plasma cell malignancy. Phase II clinical trials showed that more than 50% of MM patients fail to respond to bortezomib, the only PI currently approved for clinical use. However, the mechanisms of action and bases of individual susceptibility to PI remain largely unclear, with no reliable predictor of response identified so far. Recent evidences linking proteasome activity and Ig synthesis to susceptibility to PI suggest that the exquisite sensitivity of MM cells (MMC) to PI might be explained by an imbalance between the efficiency of the ubiquitin (Ub)-proteasome pathway and the demand for proteasome-mediated degradation. We set out to explore this hypothesis both in vitro and ex vivo. To achieve this aim, we employed human MM cell lines characterized by differential apoptotic sensitivity to PI (U266 and RPMI8226, fairly resistant cell lines, versus MM.1S, an extremely sensitive one) and primary, patient derived MMC. In MM cell lines, we found that high apoptotic sensitivity to PI is associated with lower expression of active proteasomes (as assessed by decreased expression of cleaved catalytic subunits and enzymatic assays with fluorogenic substrates in cell extracts), together with higher proteasomal workload (demonstrated by higher proteasome-dependent loss of TCA-insoluble radioactivity in pulse-chase assays). Indeed, MM.1S cells displayed 2–3 times lower proteasomal activity as compared to the more resistant U266 and RPMI8226 cells, both on a per cell basis and upon normalization by protein content. Together with the reduced proteasome capacity, MM.1S cells showed a consistently higher production of client proteins for the Ub-proteasome pathway. Such an increased load appears to be the consequence of a higher production of Rapidly Degraded Polypeptides (RDP). These are newly synthesized proteins which are quickly redirected to proteasome-mediated degradation. The imbalance between proteasomal load and capacity results in remarkable accumulation of poly-Ub proteins at the expense of free Ub (as established by both western blotting and immunofluorescence), unveiling basal proteasome stress in PI-sensitive MMC. In order to establish a causal link between proteasome stress and sensitivity to PI, we pharmacologically modulated either proteasome expression or workload and successfully altered PI-induced apoptosis. As predicted, increasing proteasome workload by means of ER stressors (e.g. tunicamycin, thapsigargin, brefeldin A) dramatically enhances susceptibility to PI, while a raise in proteasomal activity (achieved by exploiting the proteasome stress response, an adaptive mechanism by which mammalian cells induce proteasome biogenesis in response to either decreased proteasome function or increased proteasomal demand), confers marked resistance to PI-induced apoptosis. Having established cause-effect relationships between determinants of proteasome stress and vulnerability to PI in vitro, we then asked if our model could be used to predict responsiveness to PI in MM patients. In keeping with this hypothesis, intracellular immunostaining in primary, patient-derived MMC reveals that accumulation of poly-Ub proteins specifically hallmarks neoplastic plasma cells, indicating that the cancer compartment in MM patients suffers from proteasome stress. Moreover, poly-Ub levels positively correlate with Ig content, both intra- and inter-patient, suggesting a direct effect of Ig synthesis and/or retention on proteasome functional load. Finally, overall proteasome activity of primary MMC inversely correlates with the intrinsic apoptotic sensitivity to PI as assessed ex vivo, providing a rationale for the assessment of this parameter as a potential predictor of the in vivo response to bortezomib or other PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMC to PI, providing both a novel predictive tool of potential prognostic value and the framework for novel combination therapies aimed at exacerbating proteasome stress in MM.

Upon encounter with Ag, B lymphocytes undergo terminal differentiation into plasma cells, highly specialized Ab secretors that mediate humoral immune responses. Profound changes adapt cellular morphology and proteome to the new secretory functions. Although a massive secretory activity is expected to require an efficient ubiquitin-proteasome degradation system, recent in vitro studies have surprisingly revealed that the proteasome function sharply decreases during plasma cell development, thereby limiting the proteolytic capacity. We challenged this paradigm in mouse models of B cell activation, and observed that following polyclonal activation, proteasome activity decreases more than previously reported in vitro. This decrease is linked to enhanced apoptosis after treatment with the potent anti-myeloma proteasome inhibitor PS-341. Accordingly, in vivo treatment with PS-341 decreases Ab titres in T-dependent and -independent mouse immunization models. This study provides the rationale for limiting the activity of Ab-secreting cells in vivo by impacting proteasome function.

After few days of intense immunoglobulin (Ig) secretion, most plasma cells undergo apoptosis, thus ending the humoral immune response. We asked whether intrinsic factors link plasma cell lifespan to Ig secretion. Here we show that in the late phases of plasmacytic differentiation, when antibody production becomes maximal, proteasomal activity decreases. The excessive load for the reduced proteolytic capacity correlates with accumulation of polyubiquitinated proteins, stabilization of endogenous proteasomal substrates (including Xbp1s, IkappaBalpha, and Bax), onset of apoptosis, and sensitization to proteasome inhibitors (PI). These events can be reproduced by expressing Ig-mu chain in nonlymphoid cells. Our results suggest that a developmental program links plasma cell death to protein production, and help explaining the peculiar sensitivity of normal and malignant plasma cells to PI.

Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.

Many patients affected by early onset familial Alzheimer's disease (FAD), carry mutations in the presenilin 1 ( PS1 ) gene. Since it has been suggested that FAD-linked PS1 mutations impair the unfolded protein response (UPR) due to endoplasmic reticulum (ER) stress, we analyzed the UPR and amyloid β-protein processing in fibroblasts bearing various PS1 mutations. Neither in normal conditions nor after induction of ER stress with DTT or tunicamycin were the mRNA levels of UPR-responsive genes (BiP and PDI) significantly different in control and FAD fibroblasts. DTT, which blocked APP transport to the Golgi, caused a 30% decrease of secreted Aβ42 in wild type and PS1 mutant fibroblasts. In contrast, tunicamycin, which allowed exit of APP from the ER, increased secreted Aβ42 only in PS1 mutant fibroblasts. Our findings suggest that, although the UPR is active in fibroblasts from FAD patients, mutant PS1 may selectively increase Aβ42 secretion when N-glycosylation is impaired.

Immunotoxicology is the subspecialty concerned with the study of the ill effects which result from the interaction of xenobiotics with the immune system. Although these adverse effects can be the consequence of an immune reaction to the foreign substance, we shall here focus exclusively on the aspect of the damage inflicted by xenobiotics on immunity.

Macrophages (M phi diameter) from three mouse strains with genetically distinct M phi diameter deficits (C3H/HeJ, A/J, and P/J) were unable to develop high cytolytic and cytotoxic activity against tumor cells in vitro when exposed to agents (MAF and IFN-beta) that strongly increased the tumoricidal capacity of M phi diameter from nondefective C3H/HeN mice. Nevertheless, the tumoricidal deficits of M phi diameter from the defective strains did not affect their suppressive capacity on Con A-induced lymphoproliferation, nor their ability to react to IFN-beta by decreasing suppressive activity. In fact, natural suppressive activity and IFN-beta-induced changes in the suppression of M phi diameter from C3H/HeJ, A/J, and P/J mice were highly comparable to those of C3H/HeN M phi diameter, thus stressing the dissociation between the mechanisms governing M phi diameter suppression and M phi diameter tumoricidal activity. Analysis of the modulation by MAF and IFN-beta of M phi diameter ability to release the oxygen metabolites O2- and H2O2, molecules possibly involved in the effector mechanism of both M phi diameter cytotoxicity and suppression, revealed a close correlation with the patterns of suppressive activity in both nondefective and defective strains. In contrast, no correlation between the production of oxygen-reactive species and M phi diameter tumoricidal activity was observed. The ability of MAF- and IFN-beta-treated M phi diameter to produce PGE, a molecule of major importance in M phi diameter-mediated suppression and possibly involved also in the regulation of M phi diameter tumoricidal activity, again paralleled M phi diameter suppressive capacity. Thus, the mechanisms controlling M phi diameter antitumor activity appeared to be clearly distinct from those involved in M phi diameter suppression.