Estimated Prevalence and Clinical Manifestations of UBA1 Variants Associated With VEXAS Syndrome in a Clinical Population.

Importance: VEXAS (vacuoles, E1-ubiquitin-activating enzyme, X-linked, autoinflammatory, somatic) syndrome is a disease with rheumatologic and hematologic features caused by somatic variants in UBA1. Pathogenic variants are associated with a broad spectrum of clinical manifestations. Knowledge of prevalence, penetrance, and clinical characteristics of this disease have been limited by ascertainment biases based on known phenotypes. Objective: To determine the prevalence of pathogenic variants in UBA1 and associated clinical manifestations in an unselected population using a genomic ascertainment approach. Design, Setting, and Participants: This retrospective observational study evaluated UBA1 variants in exome data from 163 096 participants within the Geisinger MyCode Community Health Initiative. Clinical phenotypes were determined from Geisinger electronic health record data from January 1, 1996, to January 1, 2022. Exposures: Exome sequencing was performed. Main Outcomes and Measures: Outcome measures included prevalence of somatic UBA1 variation; presence of rheumatologic, hematologic, pulmonary, dermatologic, and other findings in individuals with somatic UBA1 variation on review of the electronic health record; review of laboratory data; bone marrow biopsy pathology analysis; and in vitro enzymatic assays. Results: In 163 096 participants (mean age, 52.8 years; 94% White; 61% women), 11 individuals harbored likely somatic variants at known pathogenic UBA1 positions, with 11 of 11 (100%) having clinical manifestations consistent with VEXAS syndrome (9 male, 2 female). A total of 5 of 11 individuals (45%) did not meet criteria for rheumatologic and/or hematologic diagnoses previously associated with VEXAS syndrome; however, all individuals had anemia (hemoglobin: mean, 7.8 g/dL; median, 7.5 g/dL), which was mostly macrocytic (10/11 [91%]) with concomitant thrombocytopenia (10/11 [91%]). Among the 11 patients identified, there was a pathogenic variant in 1 male participant prior to onset of VEXAS-related signs or symptoms and 2 female participants had disease with heterozygous variants. A previously unreported UBA1 variant (c.1861A>T; p.Ser621Cys) was found in a symptomatic patient, with in vitro data supporting a catalytic defect and pathogenicity. Together, disease-causing UBA1 variants were found in 1 in 13 591 unrelated individuals (95% CI, 1:7775-1:23 758), 1 in 4269 men older than 50 years (95% CI, 1:2319-1:7859), and 1 in 26 238 women older than 50 years (95% CI, 1:7196-1:147 669). Conclusions and Relevance: This study provides an estimate of the prevalence and a description of the clinical manifestations of UBA1 variants associated with VEXAS syndrome within a single regional health system in the US. Additional studies are needed in unselected and genetically diverse populations to better define general population prevalence and phenotypic spectrum.

RNF4-Dependent Oncogene Activation by Protein Stabilization.

Ubiquitylation regulates signaling pathways critical for cancer development and, in many cases, targets proteins for degradation. Here, we report that ubiquitylation by RNF4 stabilizes otherwise short-lived oncogenic transcription factors, including ß-catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein. RNF4 enhances the transcriptional activity of these factors, as well as Wnt- and Notch-dependent gene expression. While RNF4 is a SUMO-targeted ubiquitin ligase, protein stabilization requires the substrate's phosphorylation, rather than SUMOylation, and binding to RNF4's arginine-rich motif domain. Stabilization also involves generation of unusual polyubiquitin chains and docking of RNF4 to chromatin. Biologically, RNF4 enhances the tumor phenotype and is essential for cancer cell survival. High levels of RNF4 mRNA correlate with poor survival of a subgroup of breast cancer patients, and RNF4 protein levels are elevated in 30% of human colon adenocarcinomas. Thus, RNF4-dependent ubiquitylation translates transient phosphorylation signal(s) into long-term protein stabilization, resulting in enhanced oncoprotein activation.

Isoform-specific SCF(Fbw7) ubiquitination mediates differential regulation of PGC-1α.

The E3 ubiquitin ligase and tumor suppressor SCF(Fbw7) exists as three isoforms that govern the degradation of a host of critical cell regulators, including c-Myc, cyclin E, and PGC-1α. Peroxisome proliferator activated receptor-gamma coactivator 1α (PGC-1α) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC-1α levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin-proteasome system. Isoform-specific functions of SCF(Fbw7) are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCF(Fbw7), regulates cellular PGC-1α levels via two independent, isoform-specific, mechanisms. The cytoplasmic isoform (SCF(Fbw7ß)) reduces cellular PGC-1α levels via accelerated ubiquitin-proteasome degradation. In contrast, the nuclear isoform (SCF(Fbw7α)) increases cellular PGC-1α levels and protein stability via inhibition of ubiquitin-proteasomal degradation. When nuclear Fbw7α proteins are redirected to the cytoplasm, cellular PGC-1α protein levels are reduced through accelerated ubiquitin-proteasomal degradation. We find that SCF(Fbw7ß) catalyzes high molecular weight PGC-1α-ubiquitin conjugation, whereas SCF(Fbw7α) produces low molecular weight PGC-1α-ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC-1α levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC-1α levels via selective isoform-specific ubiquitination. Fbw7ß reduces cellular PGC-1α via ubiquitin-mediated degradation, whereas Fbw7α increases cellular PGC-1α via ubiquitin-mediated stabilization.

The Future of Children's Health in the Genomic Era.

The effects of genomic medicine on child health promise to be profound. Medical applications will eventually include characterizing patients' genomes to detect predictive mutations for pre-symptomatic counseling where treatment exists; to search for causes of diseases of unknown etiology, and to detect carriers for prenatal counseling; to define cancer and other disease-based genomes to design individualized therapy; and to understand our microbiomes to modify these in health and disease. Rapid advances in technology and bioinformatics have reduced the cost and the time and increased the accuracy necessary to sequence whole genomes or whole exomes. However, complete understanding of disease will also require correlation of genomic information with high-quality phenotypic data. In addition, several critical ethical, psycho-social, and public policy issues will require clarity in the coming years. Ultimately these advances will improve the effectiveness of health care for children and for society.

Ubiquitin proteasome-dependent degradation of the transcriptional coactivator PGC-1{alpha} via the N-terminal pathway.

PGC-1α is a potent, inducible transcriptional coactivator that exerts control on mitochondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1α levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Here, we demonstrate that PGC-1α is rapidly degraded in the nucleus (t(½ 0.3 h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, are nuclear and rapidly degraded (t(½) 0.5 h), consistent with degradation via the N terminus-dependent ubiquitin subpathway. Both PGC-1α and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally occurring N-terminal splice variant of 270 residues, NT-PGC-1α is cytoplasmic and stable (t(½>7 h), providing additional evidence that PGC-1α is degraded in the nucleus. These results strongly suggest that the nuclear N terminus-dependent ubiquitin proteasome pathway governs PGC-1α cellular degradation. In contrast, the cellular localization of NT-PCG-1α results in a longer-half-life and possible distinct temporal and potentially biological actions.

Low-density lipoprotein receptor-related protein 1 promotes cancer cell migration and invasion by inducing the expression of matrix metalloproteinases 2 and 9.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor involved in the metabolism of various extracellular ligands, including proteinases, that play critical roles in tumor invasion. Although several studies have shown an increased expression of LRP1 in cancer cells, its function in tumor development and progression remains largely unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and MMP9 and thereby promotes the migration and invasion of human glioblastoma U87 cells. Knockdown of LRP1 expression greatly decreased U87 cell migration and invasion, which was rescued by the forced expression of a functional LRP1 minireceptor. Inhibition of ligand binding to LRP1 by a specific antagonist, receptor-associated protein, also led to reduced cancer cell migration and invasion. Because MMPs play critical roles in cancer cell migration and invasion, we examined the expression of several MMPs and found that the expression of functional MMP2 and MMP9 was selectively decreased in LRP1 knockdown cells. More importantly, decreased cell migration and invasion of LRP1 knockdown cells were completely rescued by exogenous expression of MMP2 or MMP9, suggesting that these MMPs are likely downstream targets of LRP1-mediated signaling. We further show that the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased in LRP1-silenced cells, suggesting that ERK is a potential mediator of LRP1-regulated MMP2 and MMP9 expression in U87 cells. Together, our data strongly suggest that LRP1 promotes glioblastoma cell migration and invasion by regulating the expression and function of MMP2 and MMP9 perhaps via an ERK-dependent signaling pathway.

Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology.

Cellular proteins are in a dynamic state maintained by synthesis and degradation. The ubiquitin proteolytic pathway is responsible for the degradation of the bulk of cellular proteins including short-lived, regulatory, and misfolded/denatured proteins. Ubiquitin-mediated proteolysis involves covalent attachment of multiple ubiquitin molecules to the protein substrate and degradation of the targeted protein by the 26S proteasome. Recent understanding of the molecular mechanisms involved provides a framework to understand a wide variety of human pathophysiological states as well as therapeutic interventions. This review focuses on the response to hypoxia, inflammatory diseases, neurodegenerative diseases, and muscle-wasting disorders, as well as human papillomaviruses, cervical cancer and other malignancies.

Slow endocytosis of the LDL receptor-related protein 1B: implications for a novel cytoplasmic tail conformation.

The LDL receptor-related protein 1B (LRP1B) is a putative tumor suppressor homologous to LRP1. Both LRP1 and LRP1B contain cytoplasmic tails with several potential endocytosis motifs. Although the positions of these endocytic motifs are similar in both receptors, LRP1B is internalized at a 15-fold slower rate than LRP1. To determine whether the slow endocytosis of LRP1B is due to the utilization of an endocytosis motif other than the YATL motif used by LRP1, we tested minireceptors with mutations in each of the five potential motifs in the LRP1B tail. Only mutation of both NPXY motifs together abolished LRP1B endocytosis, suggesting that LRP1B can use either of these motifs for internalization. LRP1B contains a unique insertion of 33 amino acids not present in LRP1 that could lead to altered recognition of trafficking motifs. Surprisingly, deletion of this insertion had no effect on the endocytosis rate of LRP1B. However, replacing either half of the LRP1B tail with the corresponding LRP1 sequence markedly accelerated LRP1B endocytosis. From these data, we propose that both halves of the LRP1B cytoplasmic tail contribute to a unique global conformation, which results in less efficient recognition by endocytic adaptors and a slow endocytosis rate.

E12 and E47 modulate cellular localization and proteasome-mediated degradation of MyoD and Id1.

Programs of tissue differentiation are likely controlled by factors regulating gene expression and protein degradation. In muscle, the degradation of the muscle transcription factor MyoD and its inhibitor Id1 occurs via the ubiquitin-proteasome system. E12 and E47, splice products of the E2A gene, interact with MyoD to activate transcription of the muscle program and are also degraded by the ubiquitin-proteasome system (t(1/2) = approximately 6 h). E12 and E47 each contain two regions of basic amino acids, which, when mutated, lead to cytoplasmic accumulation of the proteins. These NLS mutants (E12(NLS), E47(NLS)) are degraded with a half-life similar to the wild-type proteins. In nonmuscle cells, cotransfection of either E12 or E47 with MyoD extended MyoD's half-life from approximately 1 to approximately 4 h. In addition, cotransfection of either E12 or E47 with Id1 led to a marked reduction in Id1's degradation rate from t(1/2) of approximately 1 to approximately 8 h. Furthermore, the cotransfection of NLS deficient mutants of MyoD or Id1 with E12 or E47 resulted in altered intracellular localization of the proteins largely dependent upon the E12 or E47 moiety. Cotransfection of wild-type MyoD or Id1 with NLS deficient mutants of E12 or E47 also led to an altered intracellular localization of MyoD and Id1. These results demonstrate in vivo that E12 and E47 modulate both MyoD and Id1 degradation and may have implications for the physiological regulation of muscle development.

Ubiquitin-proteasome-mediated degradation, intracellular localization, and protein synthesis of MyoD and Id1 during muscle differentiation.

Mammalian skeletal myogenesis results in the differentiation of myoblasts to mature syncytial myotubes, a process regulated by an intricate genetic network of at least three protein families: muscle regulatory factors, E proteins, and Id proteins. MyoD, a key muscle regulatory factor, and its negative regulator Id1 have both been shown to be degraded by the ubiquitin-proteasome system. Using C2C12 cells and confocal fluorescence microscopy, we showed that MyoD and Id1 co-localize within the nucleus in proliferating myoblasts. In mature myotubes, in contrast, they reside in distinctive subcellular compartments, with MyoD within the nucleus and Id1 exclusively in the cytoplasm. Cellular abundance of Id1 was markedly diminished from the very onset of muscle differentiation, whereas MyoD abundance was reduced to a much lesser extent and only at the later stages of differentiation. These reductions in MyoD and Id1 protein levels seem to result from a change in the rate of protein synthesis rather than the rate of degradation. In vivo protein stability studies revealed that the rates of ubiquitin-proteasome-mediated MyoD and Id1 degradation are independent of myogenic differentiation state. Id1 and MyoD were both rapidly degraded, each with a t 1/2 approximately = 1 h in myoblasts and in myotubes. Furthermore, relative protein synthesis rates for MyoD and Id1 were significantly diminished during myoblast to myotube differentiation. These results provide insight as to the interaction between MyoD and Id1 in the process of muscle differentiation and have implications for the involvement of the ubiquitin-proteasome-mediated protein degradation and protein synthesis in muscle differentiation and metabolism under abnormal and pathological conditions.

The ubiquitin system: pathogenesis of human diseases and drug targeting.

With the many processes and substrates targeted by the ubiquitin pathway, it is not surprising to find that aberrations in the system underlie, directly or indirectly, the pathogenesis of many diseases. While inactivation of a major enzyme such as E1 is obviously lethal, mutations in enzymes or in recognition motifs in substrates that do not affect vital pathways or that affect the involved process only partially may result in a broad array of phenotypes. Likewise, acquired changes in the activity of the system can also evolve into certain pathologies. The pathological states associated with the ubiquitin system can be classified into two groups: (a) those that result from loss of function-mutation in a ubiquitin system enzyme or in the recognition motif in the target substrate that lead to stabilization of certain proteins, and (b) those that result from gain of function-abnormal or accelerated degradation of the protein target. Studies that employ targeted inactivation of genes coding for specific ubiquitin system enzymes and substrates in animals can provide a more systematic view into the broad spectrum of pathologies that may result from aberrations in ubiquitin-mediated proteolysis. Better understanding of the processes and identification of the components involved in the degradation of key regulatory proteins will lead to the development of mechanism-based drugs that will target specifically only the involved proteins.

LRP6 expression promotes cancer cell proliferation and tumorigenesis by altering beta-catenin subcellular distribution.

The Wnt signaling pathway plays key roles in both embryogenesis and tumorigenesis. The low-density lipoprotein (LDL) receptor-related protein-6 (LRP6), a novel member of the expanding LDL receptor family, functions as an indispensable co-receptor for the Wnt signaling pathway. Although the role of LRP6 in embryonic development is now well established, its role in tumorigenesis is unclear. We report that LRP6 is readily expressed at the transcript level in several human cancer cell lines and human malignant tissues. Furthermore, using a retroviral gene transfer system, we find that stable expression of LRP6 in human fibrosarcoma HT1080 cells alters subcellular beta-catenin distribution such that the cytosolic beta-catenin level is significantly increased. This is accompanied by a significant increase in Wnt/beta-catenin signaling and cell proliferation. Finally, we demonstrate that LRP6 expression promotes tumorigenesis in vivo. These results thus indicate that LRP6 may function as a potential oncogenic protein by modulating Wnt/beta-catenin signaling.

The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system. Direct evidence for ubiquitination at the N-terminal residue.

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.

Ubiquitin-Proteasome-mediated degradation of Id1 is modulated by MyoD.

Degradation of many short-lived cellular proteins such as the transcription factor MyoD occurs via the ubiquitin-proteasome pathway. MyoD, similar to many rapidly degraded regulatory factors, interacts with several high affinity binding partners, including members of the Id (inhibitors of DNA binding) family. Following transfection to HeLa cells, Id1 is localized to the nucleus and rapidly (t(1/2) approximately 1 h) degraded via the ubiquitin-proteasome system. Mutagenesis of lysine residues within the putative nuclear localization region (amino acids 68-82) directs Id1(NLS) to the cytoplasm yet confers an increased rate of degradation (t(1/2) approximately 0.5 h). Id1 in which all lysine residues were mutagenized to alanine (lysineless Id1) was also rapidly degraded (t(1/2) approximately 0.6 h). Addition of a Myc(6) tag to the N terminus of lysine-less Id1 markedly stabilized Id1 (t(1/2) > 10 h) and suggests degradation via the N terminus-dependent pathway. Co-transfection of MyoD with Id1 or Id1(NLS) increases Id1 or Id1(NLS) within the nucleus and markedly reduces the rate of Id1 or Id1(NLS) degradation. These results thus demonstrate that in vivo MyoD modulates the rate of Id1 degradation and suggest a dynamic interplay of these factors.

Degradation of the LDL receptor class 2 mutants is mediated by a proteasome-dependent pathway.

Familial hypercholesterolemia is a genetic disorder that results from various gene mutations, primarily within the LDL receptor (LDLR). Approximately 50% of the LDLR mutations are defined as class 2 mutations, with the mutant proteins partially or entirely retained in the endoplasmic reticulum. To determine the degradation pathway of the LDLR class 2 mutants, we examined the effects of inhibition of several potential pathways on the levels of the wild-type LDLR and its four representative class 2 mutants (S156L, C176Y, E207K, and C646Y) stably expressed in Chinese hamster ovary (CHO) cells. We found that proteasome inhibitors MG132 and lactacystin blocked the degradation of the LDLR mutants, but not that of the wild-type LDLR. Treatment of CHO cells with these proteasome inhibitors led to a significant accumulation of the mutants at steady state. Furthermore, cell surface levels of the LDLR mutants were significantly increased upon inhibition of the proteasome degradation pathway. In contrast to the proteasome inhibitors, inhibitors of trypsin-like proteases, chymotrypsin-like proteases, and lysosomal pathway inhibitors did not affect the levels of the LDLR mutants. Taken together, these data demonstrate that the proteasome is the principal degradation pathway for LDLR class 2 mutants.

Degradation of the Id2 developmental regulator: targeting via N-terminal ubiquitination.

Degradation of cellular proteins via the ubiquitin-proteasome system (UPS) involves: (i) generation of a substrate-anchored polyubiquitin degradation signal and (ii) destruction of the tagged protein by the 26S proteasome with release of free and reusable ubiquitin. For most substrates, it is believed that the first ubiquitin moiety is conjugated to a epsilon-NH(2) group of an internal Lys residue. Recent findings indicate that for several proteins, the first ubiquitin moiety is fused, in a linear manner, to the free alpha-NH(2) group of the protein. Here, we demonstrate that the inhibitor of differentiation (or inhibitor of DNA binding) 2, Id2, that downregulates gene expression in undifferentiated and self-renewing cells, is degraded by the UPS following ubiquitination at its N-terminal residue. Lysine-less (LL) Id2 is degraded efficiently by the proteasome following ubiquitination. Fusion of a Myc tag to the N-terminal but not to the C-terminal residue of Id2 stabilizes the protein. Furthermore, deletion of the first 15 N-terminal residues of Id2 stabilizes the protein, suggesting that this domain serves as a recognition element, possibly for the ubiquitin ligase, E3. The mechanisms and structural motives that govern Id2 stability may have important implications to the regulation of the protein during normal differentiation and malignant transformation.

Proteasome regulates the delivery of LDL receptor-related protein into the degradation pathway.

The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin-proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway.

Low density lipoprotein (LDL) receptor-related protein 1B impairs urokinase receptor regeneration on the cell surface and inhibits cell migration.

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA.PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.

Receptor-associated protein facilitates proper folding and maturation of the low-density lipoprotein receptor and its class 2 mutants.

Familial hypercholesterolemia is the consequence of various mutations in the low-density lipoprotein receptor (LDLR). In the current study, we show that a specialized molecular chaperone, the receptor-associated protein (RAP), promotes proper folding and subsequent exocytic trafficking of the wild-type LDLR and several of its class 2 mutants. Co-immunoprecipitation with anti-RAP antibody demonstrates that RAP interacts with the LDLR. Kinetic analyses of LDLR posttranslational folding and maturation in the absence or presence of RAP coexpression show that RAP prevents aggregation and promotes the maturation of the LDLR. Additionally, depletion of Ca(2+) in intact cells impairs LDLR folding, and coexpression of RAP partially corrects this misfolding. Finally, we show that the increased mature cell surface LDLR in the presence of RAP coexpression is functional in its ability to endocytose and degrade (125)I-LDL. Taken together, our results show that the folding, trafficking, and maturation of the LDLR and its class 2 mutants are promoted by RAP.