Endocytosis Of Membrane Proteins Research Articles

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

DNA-functionalized artificial mechanoreceptor for de novo force-responsive signaling.

Synthetic signaling receptors enable programmable cellular responses coupling with customized inputs. However, engineering a designer force-sensing receptor to rewire mechanotransduction remains largely unexplored. Herein, we introduce nongenetically engineered artificial mechanoreceptors (AMRs) capable of reprogramming non-mechanoresponsive receptor tyrosine kinases (RTKs) to sense user-defined force cues, enabling de novo-designed mechanotransduction. AMR is a modular DNA-protein chimera comprising a mechanosensing-and-transmitting DNA nanodevice grafted on natural RTKs via aptameric anchors. AMR senses intercellular tensile force via an allosteric DNA mechano-switch with tunable piconewton-sensitive force tolerance, actuating a force-triggered dynamic DNA assembly to manipulate RTK dimerization and activate intracellular signaling. By swapping the force-reception ligands, we demonstrate the AMR-mediated activation of c-Met, a representative RTK, in response to the cellular tensile forces mediated by cell-adhesion proteins (integrin, E-cadherin) or membrane protein endocytosis (CI-M6PR). Moreover, AMR also allows the reprogramming of FGFR1, another RTK, to customize mechanobiological function, for example, adhesion-mediated neural stem cell maintenance.

Local endocytosis of sucrose transporter 2 in duckweed reveals the role of sucrose transporter 2 in guard cells.

The local endocytosis of membrane proteins is critical for many physiological processes in plants, including the regulation of growth, development, nutrient absorption, and osmotic stress response. Much of our knowledge on the local endocytosis of plasma membrane (PM) protein only focuses on the polar growth of pollen tubes in plants and neuronal axon in animals. However, the role of local endocytosis of PM proteins in guard cells has not yet been researched. Here, we first cloned duckweed SUT2 (sucrose transporter 2) protein and then conducted subcellular and histological localization of the protein. Our results indicated that LpSUT2 (Landoltia punctata 0202 SUT2) is a PM protein highly expressed on guard cells. In vitro experiments on WT (wild type) lines treated with high sucrose concentration showed that the content of ROS (reactive oxygen species) in guard cells increased and stomatal conductance decreased. We observed the same results in the lines after overexpression of the LpSUT2 gene with newfound local endocytosis of LpSUT2. The local endocytosis mainly showed that LpSUT2 was uniformly distributed on the PM of guard cells in the early stage of development, and was only distributed in the endomembrane of guard cells in the mature stage. Therefore, we found the phenomenon of guard cell LpSUT2 local endocytosis through the changes of duckweed stomata and concluded that LpSUT2 local endocytosis might be dependent on ROS accumulation in the development of duckweed guard cells. This paper might provide future references for the genetic improvement and water-use efficiency in other crops.

A phosphorylation network controls the stability of alpha‐arrestins Aly1/Art6 and Aly2/Art3

Selective protein trafficking controls the repertoire of membrane proteins at the cell surface. This contingent of surface proteins regulates nutrient/metabolite balance and response to extracellular cues. Environmental changes trigger cellular signaling that drives transitions in the plasma membrane proteome. These alterations are achieved in no small part through regulation of the α‐arrestins, an emergent and powerful class of protein trafficking adaptors. The α‐arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. α‐Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of α‐arrestins is key to regulating their trafficking function; dephosphorylated α‐arrestins are generally ‘active’ endocytic adaptors. While some kinases and phosphatases for α‐arrestins are known, the degree of α‐arrestins phosphorylation is staggering, with >40 phosphorylation sites identified for a single α‐arrestin, and suggests we still have much to learn about α‐arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous α‐arrestins Aly1 and Aly2 using a genetic screen in yeast. Using an array of all known non‐essential kinase and phosphatase deletions, we determined which of these influenced α‐arrestin‐mediated resistance to rapamycin, an inhibitor of the TORC1 nutrient‐sensing kinase. We identified a large cohort of kinases and phosphates that influenced Aly‐dependent phenotypes, causing electrophoretic mobility changes and, in many cases, diminishing the abundance of these α‐arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other α‐arrestins, identified in our screen. Strikingly, Aly1 and Aly2 were hyperphosphorylated and destabilized in the absence of Sit4, suggesting that excessive phosphorylation may promote degradation of these α‐arrestins. For Aly2, but not Aly1, degradation in the sit4∆ cells could be reversed by loss of the vacuolar protease Pep4, indicating that vacuolar degradation predominates for Aly2 under these conditions. Loss of the Npr1 kinase in sit4∆ cells restored Aly protein abundances and reversed the hyperphosphorylation, demonstrating that this kinase is responsible for the excess Aly phosphorylation in sit4∆ cells. This is a remarkable finding as typically Npr1 is considered inactive in sit4∆, however, we suggest that Npr1 is selectively active in the absence of Sit4, able to modify some substrates but not others. We define new features of TORC1 signaling in regulating α‐arrestin phosphorylation and stability.

MS4A3 Promotes Differentiation in Chronic Myeloid Leukemia By Enhancing Common β Chain Cytokine Receptor Endocytosis

MS4A3 Promotes Differentiation in Chronic Myeloid Leukemia By Enhancing Common β Chain Cytokine Receptor Endocytosis

Nicastrin-Like, a Novel Transmembrane Protein from Trypanosoma cruzi Associated to the Flagellar Pocket

Nicastrin (NICT) is a transmembrane protein physically associated with the polytypical aspartyl protease presenilin that plays a vital role in the correct localization and stabilization of presenilin to the membrane-bound γ-secretase complex. This complex is involved in the regulation of a wide range of cellular events, including cell signaling and the regulation of endocytosed membrane proteins for their trafficking and protein processing. Methods: In Trypanosoma cruzi, the causal agent of the Chagas disease, a NICT-like protein (Tc/NICT) was identified with a short C-terminus orthologous to the human protein, a large ectodomain (ECD) with numerous glycosylation sites and a single-core transmembrane domain containing a putative TM-domain (457GSVGA461) important for the γ-secretase complex activity. Results: Using the Spot-synthesis strategy with Chagasic patient sera, five extracellular epitopes were identified and synthetic forms were used to generate rabbit anti-Tc/NICT polyclonal serum that recognized a ~72-kDa molecule in immunoblots of T. cruzi epimastigote extracts. Confocal microscopy suggests that Tc/NICT is localized in the flagellar pocket, which is consistent with data from our previous studies with a T. cruzi presenilin-like protein. Phylogenetically, Tc/NICT was localized within a subgroup with the T. rangeli protein that is clearly detached from the other Trypanosomatidae, such as T. brucei. These results, together with a comparative analysis of the selected peptide sequence regions between the T. cruzi and mammalian proteins, suggest a divergence from the human NICT that might be relevant to Chagas disease pathology. As a whole, our data show that a NICT-like protein is expressed in the infective and replicative stages of T. cruzi and may be considered further evidence for a γ-secretase complex in trypanosomatids.

Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies

A safe and controlled manipulation of endocytosis invivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the invivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor exvivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters.

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM–endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.

Single Event Resolution of Plant Plasma Membrane Protein Endocytosis by TIRF Microscopy.

Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, nutrient delivery, toxin avoidance, and pathogen defense. The precise mechanisms of endocytosis in plants remain quite elusive. The lack of direct visualization and examination of single events of endocytosis has greatly hampered our ability to precisely monitor the cell surface lifetime and the recruitment profile of proteins driving endocytosis or endocytosed cargos in plants. Here, we discuss the necessity to systematically implement total internal reflection fluorescence microcopy (TIRF) in the Plant Cell Biology community and present reliable protocols for high spatial and temporal imaging of endocytosis in plants using clathrin-mediated endocytosis as a test case, since it represents the major route for internalization of cell-surface proteins in plants. We developed a robust method to directly visualize cell surface proteins using TIRF microscopy combined to a high throughput, automated and unbiased analysis pipeline to determine the temporal recruitment profile of proteins to single sites of endocytosis, using the departure of clathrin as a physiological reference for scission. Using this ‘departure assay’, we assessed the recruitment of two different AP-2 subunits, alpha and mu, to the sites of endocytosis and found that AP2A1 was recruited in concert with clathrin, while AP2M was not. This validated approach therefore offers a powerful solution to better characterize the plant endocytic machinery and the dynamics of one’s favorite cargo protein.

Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells.

Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment.

Basigin-mediated redistribution of CD98 promotes cell spreading and tumorigenicity in hepatocellular carcinoma.

BackgroundDysregulated endocytosis of membrane proteins contributes significantly to several hallmarks of cancer. Basigin can enhance cancer progression, but its precise mechanism remains unclear. CD98 promotes cell spreading and tumorigenicity by triggering integrin clustering and enhancing cell adhesion to the extracellular matrix. The endocytosis and recyle of basigin and CD98 might play critical roles in cancer.MethodsThe role of CD98 was confirmed in liver cancer cells by cell spreading in vitro and tumorigenicity by nude mice xenograft tumor assay in vivo; membrane expression of basigin and CD98 in SMMC-7721 was measured by FCAS; pull down and SPR analysis were uses to reveal the direct association between basigin and CD98; DsRed1 tagged CD98 was blocked in the cytoplasm in K7721 (whose basigin was knockn out) and had a well colocalization with ER and Rab5a positive recycling endosomes under co-focal; finally, by FRET imaging and FCAS we observed the internalization of basigin and CD98 was flotillin-1-regulated, and their recycle at early steps was Arf6-mediated.ResultsBasigin and CD98 were highly expressed and co-localized on the human hepatocellular carcinoma (HCC) cell membrane; basigin can directly bind to CD98, mediating CD98 redistribution on the HCC cell membrane and activating the downstream integrin signaling pathway. Internalization of basigin and CD98 was flotillin-1 regulated the and their recycling was mediated by Arf6. This recycling process for basigin and CD98 promotes cell spreading and tumor growth in liver cancer xenografts.ConclusionBasigin, as a redistribution chaperone of CD98, plays a critical role in promoting cell spreading and the progression of hepatocellular carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0226-6) contains supplementary material, which is available to authorized users.

A Histidine-rich Linker Region in Peptidylglycine α-Amidating Monooxygenase Has the Properties of a pH Sensor

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

Gamma-secretase subunits associate in intracellular membrane compartments in Arabidopsis thaliana.

Gamma-secretase is a multisubunit complex with intramembrane proteolytic activity. In humans it was identified in genetic screens of patients suffering from familial forms of Alzheimer's disease, and since then it was shown to mediate cleavage of more than 80 substrates, including amyloid precursor protein or Notch receptor. Moreover, in animals, γ-secretase was shown to be involved in regulation of a wide range of cellular events, including cell signalling, regulation of endocytosis of membrane proteins, their trafficking, and degradation. Here we show that genes coding for γ-secretase homologues are present in plant genomes. Also, amino acid motifs crucial for γ-secretase activity are conserved in plants. Moreover, all γ-secretase subunits: PS1/PS2, APH-1, PEN-2, and NCT colocalize and interact with each other in Arabidopsis thaliana protoplasts. The intracellular localization of γ-secretase subunits in Arabidopsis protoplasts revealed a distribution in endomembrane system compartments that is consistent with data from animal studies. Together, our data may be considered as a starting point for analysis of γ-secretase in plants.

A Calcineurin-dependent Switch Controls the Trafficking Function of α-Arrestin Aly1/Art6

Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.

Single-Axonal Organelle Analysis Method Reveals New Protein–Motor Associations

Axonal transport of synaptic vesicle proteins is required to maintain neurons' ability to communicate via synaptic transmission. Neurotransmitter-containing synaptic vesicles are assembled at synaptic terminals via highly regulated endocytosis of membrane proteins. These synaptic vesicle membrane proteins are synthesized in the cell body and transported to synapses in carrier vesicles that make their way down axons via microtubule-based transport utilizing kinesin molecular motors. Identifying the cargos that each kinesin motor protein carries from the cell bodies to the synapse is key to understanding both diseases caused by motor protein dysfunction and how synaptic vesicles are assembled. However, obtaining a bulk sample of axonal transport complexes from central nervous system (CNS) neurons to use for identification of their contents has posed a challenge to researchers. To obtain axonal carrier vesicles from primary cultured neurons, we fabricated a microfluidic chip designed to physically isolate axons from dendrites and cell bodies and developed a method to remove bulk axonal samples and label their contents. Synaptic vesicle protein carrier vesicles in these samples were labeled with antibodies to the synaptic vesicle proteins p38, SV2A, and VAMP2, and the anterograde axonal transport motor KIF1A, after which antibody overlap was evaluated using single-organelle TIRF microscopy. This work confirms a previously discovered association between KIF1A and p38 and shows that KIF1A also transports SV2A- and VAMP2-containing carrier vesicles.

The Scribble polarity protein stabilizes E-cadherin/p120-catenin binding and blocks retrieval of E-cadherin to the Golgi.

Several polarity proteins, including Scribble (Scrb) have been implicated in control of vesicle traffic, and in particular the endocytosis of E-cadherin, but through unknown mechanisms. We now show that depletion of Scrb enhances endocytosis of E-cadherin by weakening the E-cadherin-p120catenin interaction. Unexpectedly, however, the internalized E-cadherin is not degraded but accumulates in the Golgi apparatus. Silencing p120-catenin causes degradation of E-cadherin in lysosomes, but degradation is blocked by the co-depletion of Scrb, which diverts the internalized E-cadherin to the Golgi. Loss of Scrb also enhances E-cadherin binding to retromer components, and retromer is required for Golgi accumulation of Scrb, and E-cadherin stability. These data identify a novel and unanticipated function for Scrb in blocking retromer-mediated diversion of E-cadherin to the Golgi. They provide evidence that polarity proteins can modify the intracellular itinerary for endocytosed membrane proteins.

Spatiotemporal Regulation of the Ubiquitinated Cargo-binding Activity of Rabex-5 in the Endocytic Pathway

The regulatory mechanism underlying the interaction of the Rabex-5 MIU domain with ubiquitinated cargos remains unclear. Rabex-5 guanine nucleotide exchange factor (GEF) mutants affected interactions of ubiquitinated cargos. GDP/GTP exchange in the GEF domain controls the MIU domain interactions with the ubiquitinated cargos. Rabex-5 GEF activity acts as an intramolecular switch for spatiotemporal trafficking of the ubiquitinated cargos. Ubiquitin (Ub)-dependent endocytosis of membrane proteins requires precise molecular recognition of ubiquitinated cargo by Ub-binding proteins (UBPs). Many UBPs are often themselves monoubiquitinated, a mechanism referred to as coupled monoubiquitination, which prevents them from binding in trans to the ubiquitinated cargo. However, the spatiotemporal regulatory mechanism underlying the interaction of UBPs with the ubiquitinated cargo, via their Ub-binding domains (UBDs) remains unclear. Previously, we reported the interaction of Rabex-5, a UBP and guanine nucleotide exchange factor (GEF) for Rab5, with ubiquitinated neural cell adhesion molecule L1, via its motif interacting with Ub (MIU) domain. This interaction is critical for the internalization and sorting of the ubiquitinated L1 into endosomal/lysosomal compartments. The present study demonstrated that the interaction of Rabex-5 with Rab5 depends specifically on interaction of the MIU domain with the ubiquitinated L1 to drive its internalization. Notably, impaired GEF mutants and the Rabex-5(E213A) mutant increased the flexibility of the hinge region in the HB-VPS9 tandem domain, which significantly affected their interactions with the ubiquitinated L1. In addition, GEF mutants increased the catalytic efficiency, which resulted in a reduced interaction with the ubiquitinated L1. Furthermore, the coupled monoubiquitination status of Rabex-5 was found to be significantly associated with interaction of Rabex-5 and the ubiquitinated L1. Collectively, our study reveals a novel mechanism, wherein the GEF activity of Rabex-5 acts as an intramolecular switch orchestrating ubiquitinated cargo-binding activity and coupled monoubiquitination to permit the spatiotemporal dynamic exchange of the ubiquitinated cargos.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

Protein Ubiquitylation in Pancreatic Cancer

Pancreatic cancer is one of the worst, as almost 100% of patients will die within 5 years after diagnosis. The tumors are characterized by an early, invasive, and metastatic phenotype, and extreme resistance to all known anticancer therapies. Therefore, there is an urgent need to develop new investigative strategies in order to identify new molecular targets and, possibly, new drugs to fight this disease efficiently. Whereas it has been known for more than 3 decades now, ubiquitylation is a post-translational modification of protein that only recently emerged as a major regulator of many biological functions, dependent and independent on the proteasome, whose failure is involved in many human diseases, including cancer. Indeed, despite its role in promoting protein degradation through the proteasome, ubiquitylation is now known to regulate diverse cellular processes, such as membrane protein endocytosis and intracellular trafficking, assembly of protein complexes, gene transcription, and activation or inactivation of enzymes. Taking into account that ubiquitylation machinery is a three-step process involving hundreds of proteins, which is countered by numerous ubiquitin hydrolases, and that the function of ubiquitylation relies on the recognition of the ubiquitin signals by hundreds of proteins containing a ubiquitin binding domain (including the proteasome), the number of possible therapeutic targets is exceptionally vast and will need to be explored carefully for each disease. In the case of pancreatic cancer, the study and the identification of specific alteration(s) in protein ubiquitylation may help to explain its severity and may furnish more specific targets for more efficient therapies.

The Ubiquitin-interacting Motif Protein, S5a, Is Ubiquitinated by All Types of Ubiquitin Ligases by a Mechanism Different from Typical Substrate Recognition

S5a/Rpn10 is a ubiquitin (Ub)-binding protein that is a subunit of the 26S proteasome but also exists free in the cytosol. It binds poly-Ub chains through its two Ub-interacting motifs (UIMs). We discovered that, unlike typical substrates of Ub ligases (E3s), S5a can be ubiquitinated by all E3s tested including multimeric and monomeric Ring finger E3s (MuRF1, Siah2, Parkin, APC, and SCF(betaTRCP1)), the U-box E3, CHIP, and HECT domain E3s (E6AP and Nedd4) when assayed with UbcH5 or related Ub-conjugating enzymes. However, the E2s, UbcH1 and UbcH13/Uev1a, which function by distinct mechanisms, do not support S5a ubiquitination. Thus, S5a can be used for assay of probably all E3s with UbcH5. Ubiquitination of S5a results from its binding to Ub chains on the E3 (after self-ubiquitination) or on the substrate, as a mutant lacking the UIM domain was not ubiquitinated. Furthermore, if the S5a UIM domains were fused to GST, the protein was rapidly ubiquitinated by MuRF1 and CHIP. In addition, polyubiquitination (but not monoubiquitination) of MuRF1 allowed S5a to bind to MuRF1 and accelerated S5a ubiquitination. This tendency of S5a to associate with the growing Ub chain can explain how S5a, unlike typical substrates, which are recognized by certain E3s through specific motifs, is ubiquitinated by all E3s tested and is rapidly degraded in vivo.