Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.

Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.

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An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽

10.1182/blood.v108.11.4034.4034 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4034-4034

Author(s):

Lingxia Chen ◽

Juan Li ◽

Hui Lu ◽

Haiyan Jiang ◽

Rita Sarkar ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Heavy Chain ◽

Light Chain ◽

Hek293 Cells ◽

Light Chains ◽

Trans Splicing ◽

In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

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ERdj3, a Stress-inducible Endoplasmic Reticulum DnaJ Homologue, Serves as a CoFactor for BiP's Interactions with Unfolded Substrates

Molecular Biology of the Cell ◽

10.1091/mbc.e04-05-0434 ◽

2005 ◽

Vol 16 (1) ◽

pp. 40-50 ◽

Cited By ~ 118

Author(s):

Ying Shen ◽

Linda M. Hendershot

Keyword(s):

Endoplasmic Reticulum ◽

Protein Folding ◽

Atpase Activity ◽

Light Chain ◽

Light Chains ◽

Nonpermissive Temperature ◽

Protein Substrates ◽

Unfolded Protein

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.

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IgG1 Thioether Bond Formation in Vivo

Journal of Biological Chemistry ◽

10.1074/jbc.m113.468397 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16371-16382 ◽

Cited By ~ 34

Author(s):

Qingchun Zhang ◽

Matthew R. Schenauer ◽

John D. McCarter ◽

Gregory C. Flynn

Keyword(s):

Light Chain ◽

Reaction Rate ◽

Human Subjects ◽

Light Chains ◽

Chain Reaction ◽

Healthy Human ◽

Conversion Rates ◽

Healthy Human Subjects

During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1κ therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing λ light chains than those containing κ light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with λ light chains, which may help explain the observed reaction rate differences.

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BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2209 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2209-2219 ◽

Cited By ~ 129

Author(s):

Young-Kwang Lee ◽

Joseph W. Brewer ◽

Rachel Hellman ◽

Linda M. Hendershot

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Protein Complexes ◽

Critical Role ◽

Light Chains ◽

Heavy Chains ◽

Chain Complexes ◽

Chain Synthesis

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

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Glomerular and tubular handling of differently charged human immunoglobulin light chains by the rat kidney

Clinical Science ◽

10.1042/cs0740639 ◽

1988 ◽

Vol 74 (6) ◽

pp. 639-644 ◽

Cited By ~ 10

Author(s):

Christine Baylis ◽

James Falconer-Smith ◽

Brian Ross

Keyword(s):

Light Chain ◽

Rat Kidney ◽

Tubular Reabsorption ◽

High Rate ◽

Light Chains ◽

Renal Handling ◽

Charge Dependence ◽

Intact Kidney

1. To examine the effect of charge and molecular size on the renal handling of immunoglobulin light chain, three different human light chains were purified, iodinated and characterized with regard to molecular weight and isoelectric point. The molecular weights were similar, whereas the isoelectric points ranged from markedly cationic to markedly anionic. 2. Renal handling of the light chains was determined in vivo in the Munich–Wistar rat and in vitro in the isolated perfused rat kidney. 3. Significant restriction to the transglomerular passage of all three light chains was evident in the intact kidney, with the most anionic light chain being restricted most while the cationic light chain was restricted least. The charge dependence of filtration of these naturally occurring proteins was, however, much less pronounced than has previously been reported for the synthetic dextrans. 4. Tubular reabsorption of the three light chains was quite variable in the intact kidney and did not appear to be related to molecular charge. In the isolated perfused kidney, once account had been taken of the abnormally high rate of filtration of these proteins, the reabsorption of the three light chains was strikingly similar to that seen in vivo. 5. With these three purified human light chains no marked influence of charge (pI) has been demonstrated in vivo or in vitro, for glomerular restriction or renal tubular reabsorption. Some other properties of human myeloma light chains may determine their renal handling and nephrotoxicity.

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Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649203 ◽

1977 ◽

Vol 37 (01) ◽

pp. 073-080 ◽

Cited By ~ 5

Author(s):

Knut Gjesdal ◽

Duncan S. Pepper

Keyword(s):

Macaca Mulatta ◽

Half Life ◽

Human Platelet ◽

Gel Filtration ◽

Platelet Factor 4 ◽

Active Protein ◽

Platelet Factor ◽

Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

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An epilepsy-causing mutation leads to co-translational misfolding of the Kv7.2 channel

BMC Biology ◽

10.1186/s12915-021-01040-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Janire Urrutia ◽

Alejandra Aguado ◽

Carolina Gomis-Perez ◽

Arantza Muguruza-Montero ◽

Oscar R. Ballesteros ◽

...

Keyword(s):

Structural Instability ◽

Binding Assays ◽

Iq Motif ◽

Channel Function ◽

Human Epilepsy ◽

Pathogenic Variants ◽

In Silico Studies ◽

Nascent Chain

Abstract Background The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. Results We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. Conclusions Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitro.

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68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Pharmaceutics ◽

10.3390/pharmaceutics13081160 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1160

Author(s):

Adrien Chastel ◽

Delphine Vimont ◽

Stephane Claverol ◽

Marion Zerna ◽

Sacha Bodin ◽

...

Keyword(s):

Calcium Mobilization ◽

Pharmacological Characterization ◽

Peptide Receptor ◽

Gastrin Releasing Peptide ◽

Production Route ◽

Membrane Bound ◽

One Year ◽

Calcium Mobilization Assay

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment.

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Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

International Journal of Molecular Sciences ◽

10.3390/ijms22137232 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7232

Author(s):

Gloria Lazzeri ◽

Carla L. Busceti ◽

Francesca Biagioni ◽

Cinzia Fabrizi ◽

Gabriele Morucci ◽

...

Keyword(s):

Plasma Membrane ◽

Light Chain ◽

Adrenergic Receptors ◽

Cell Damage ◽

Protective Effects ◽

Autophagy Flux ◽

Brain Areas ◽

Indirect Consequence

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane β2-adrenergic receptors (ARs). Evidence indicates that β2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

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In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3868 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3868-3872 ◽

Cited By ~ 15

Author(s):

C M Shumard ◽

C Torres ◽

D C Eichler

Keyword(s):

18S Rrna ◽

Precise Determination ◽

In Vivo Studies ◽

Rrna Sequence ◽

S1 Nuclease ◽

In Vitro Processing ◽

Rrna Maturation ◽

A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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