Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to ...activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.
•CALR mutants rescue cell surface localization of traffic-deficient TpoR, including R102P that causes congenital thrombocytopenia.•Oncogenic TpoR activation by CALR mutants requires interaction, stabilization, and cell surface localization of the TpoR-CALR complex.
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After synthesis, proteins are folded into their native conformations aided by molecular chaperones. Dysfunction in folding caused by genetic mutations in numerous genes causes protein conformational ...diseases. Membrane proteins are more prone to misfolding due to their more intricate folding than soluble proteins. Misfolded proteins are detected by the cellular quality control systems, especially in the endoplasmic reticulum, and proteins may be retained there for eventual degradation by the ubiquitin-proteasome system or through autophagy. Some misfolded proteins aggregate, leading to pathologies in numerous neurological diseases. In vitro, modulating mutant protein folding by altering molecular chaperone expression can ameliorate some misfolding. Some small molecules known as chemical chaperones also correct mutant protein misfolding in vitro and in vivo. However, due to their lack of specificity, their potential as therapeutics is limited. Another class of compounds, known as pharmacological chaperones (pharmacoperones), binds with high specificity to misfolded proteins, either as enzyme substrates or receptor ligands, leading to decreased folding energy barriers and correction of the misfolding. Because many of the misfolded proteins are misrouted but do not have defects in function per se, pharmacoperones have promising potential in advancing to the clinic as therapeutics, since correcting routing may ameliorate the underlying mechanism of disease. This review will comprehensively summarize this exciting area of research, surveying the literature from in vitro studies in cell lines to transgenic animal models and clinical trials in several protein misfolding diseases.
Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the ...binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aβ antibody, which uses a monovalent binding mode to the TfR, increases β-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.
Although the majority of low-grade, early-stage endometrial cancer patients have good survival with surgery alone, patients who recur tend to do poorly. Identification of patients at high risk of ...recurrence who would benefit from adjuvant treatment or more extensive surgical staging would help optimize individualized care of endometrial cancer patients. CTNNB1 (encodes β-catenin) mutations identify a subset of low-grade, early-stage endometrial cancer patients at high risk of recurrence. Mutation of CTNNB1 exon 3 is classically associated with translocation of the β-catenin protein from the membrane to the nucleus and activation of Wnt/β-catenin signaling. Given the clinical utility of identifying endometrial carcinomas with CTNNB1 mutation, the purpose of this study was to determine if immunohistochemistry could act as a surrogate for CTNNB1 gene sequencing. Next-generation sequencing was performed on 345 endometrial carcinomas. Immunohistochemical localization of β-catenin was determined for 53/63 CTNNB1 exon 3 mutant tumors for which tissue was available and a subset of wild-type tumors. Nuclear localization of β-catenin had 100% specificity in distinguishing CTNNB1 mutant from wild type, but sensitivity was lower (84.9%). Nearly half of CTNNB1 mutant cases had only 5–10% of tumor cells with β-catenin nuclear localization. The concordance between pathologists blinded to mutation status in assessing nuclear localization was 100%. The extent of β-catenin nuclear localization was not associated with specific CTNNB1 gene mutation, tumor grade, presence of non-endometrioid component, or specific concurrent gene mutations in the tumor. For comparison, nuclear localization of β-catenin was more diffuse in desmoid fibromatosis, a tumor also associated with CTNNB1 mutation. Thus, nuclear localization of β-catenin assessed by immunohistochemistry does not detect all endometrial cancers with CTNNB1 gene mutation. The extent of nuclear localization may be tumor type dependent. For endometrial cancer, immunohistochemistry could be an initial screen, with CTNNB1 sequencing employed when nuclear localization of β-catenin is absent.
A histidine‐rich endosomal escape peptide, as described by Juanjuan Du et al. in their Research Article (e202304692), is capable of mediating efficient endosome‐to‐cytosol transport of ...peptide/protein cargo, depicted in the cover picture as butterflies escaping from bubbles trapping them. Once combined with antibody‐mediated endocytosis, this strategy enables cell‐specific cytosolic delivery of functional proteins.
Newly synthesized transmembrane proteins undergo a series of steps to ensure that only the required amount of correctly folded protein is localized to the membrane. The regulation of protein quality ...and its abundance at the membrane are often controlled by ubiquitination, a multistep enzymatic process that results in the attachment of ubiquitin, or chains of ubiquitin to the target protein. Protein ubiquitination acts as a signal for sorting, trafficking, and the removal of membrane proteins via endocytosis, a process through which multiple ubiquitin ligases are known to specifically regulate the functions of a number of ion channels, transporters, and signaling receptors. Endocytic removal of these proteins through ubiquitin-dependent endocytosis provides a way to rapidly downregulate the physiological outcomes, and defects in such controls are directly linked to human pathologies. Recent evidence suggests that ubiquitination is also involved in the shedding of membranes and associated proteins as extracellular vesicles, thereby not only controlling the cell surface levels of some membrane proteins, but also their potential transport to neighboring cells. In this review, we summarize the mechanisms and functions of ubiquitination of membrane proteins and provide specific examples of ubiquitin-dependent regulation of membrane proteins.
Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is ...linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.
The structure–function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian ...scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular mechanism by which these membrane-active peptides lyse membranes. The last class of membrane-active peptides discussed are the CPPs, which translocate across the lipid bilayer without inducing severe disruption and have potential as drug vehicles. CPPs are typically highly charged and can show antimicrobial activity by targeting an intracellular target rather than via a direct membrane lytic mechanism. A critical aspect in the structure–function relationship of membrane-active peptides is their specific activity relative to the lipid membrane composition of the cell target. Cell membranes have a wide diversity of lipids, and those of eukaryotic and prokaryotic species differ greatly in composition and structure. The activity of AMPs from Australian tree frogs, toxins, and CPPs has been investigated within various lipid systems to assess whether a relationship between peptide and membrane composition could be identified. NMR spectroscopy techniques are being used to gain atomistic details of how these membrane-active peptides interact with model membranes and cells, and in particular, competitive assays demonstrate the difference between affinity and activity for a specific lipid environment. Overall, the interactions between these relatively small sized peptides and various lipid bilayers give insight into how these peptides function at the membrane interface.
Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional ...endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis . Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.
Significance Macroautophagy (hereafter as autophagy) involves the delivery of cytosolic materials via autophagosome upon its fusion with the endosome and lysosome/vacuole. The endosomal sorting complex required for transport (ESCRT) machinery is responsible for the formation of intraluminal vesicles (ILVs) in multivesicular bodies (MVBs) and the sorting of ubiquitinated membrane cargos into MVB ILVs for degradation. Here, we show that, in addition to regulating MVB biogenesis, the plant-specific ESCRT component FYVE domain protein required for endosomal sorting 1 (FREE1) also plays dual roles in vacuolar protein transport and autophagic degradation. FREE1 directly interacts with a plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 to manipulate the autophagic degradation in plants. Thus, we demonstrate multiple functions of FREE1 and a direct link between the ESCRT machinery and autophagy process in plants.
Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin ...tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow-derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.
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