scholarly journals Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

2021 ◽  
Author(s):  
Ritaban Halder ◽  
Daniel A. Nissley ◽  
Ian Sitarik ◽  
Edward P. O’Brien
Keyword(s):  
Tertiary Structure ◽  
Molecular Level ◽  
Meta Analysis ◽  
Hydrophobic Surface ◽  
Misfolded Proteins ◽  
Similar Degree ◽  
Functional Protein ◽  
Folded Proteins ◽  
And Function

ABSTRACTSubpopulations of soluble, misfolded proteins can bypass chaperones within cells. The scope of this phenomenon and the lifetimes of these states have not been experimentally quantified, and how such misfolding happens at the molecular level is poorly understood. We address the first issue through a meta-analysis of the experimental literature. We find that in all quantitative protein refolding-function studies, there is always a subpopulation of soluble but misfolded and less-functional protein that does not fold in the presence of one or more chaperones. This subpopulation ranges from 8% to 50% of the soluble protein molecules in solution. Fitting the experimental time traces to a kinetic model, we find these chaperone-bypassing misfolded states take months or longer to fold and function in the presence of different chaperones. We next addressed how, at the molecular level, some misfolded proteins can evade chaperones by simulating six different proteins interacting with E. coli’s GroEL and HtpG chaperones when those proteins are in folded, unfolded, or long-lived, soluble, misfolded states. We observe that both chaperones strongly bind the unfolded state and weakly bind the folded and misfolded states to a similar degree. Thus, these chaperones cannot distinguish between the folded and long-lived misfolded states of these proteins. A structural analysis reveals the misfolded states are highly similar to the native state – having a similar size, amount of exposed hydrophobic surface area, and level of tertiary structure formation. These results demonstrate that in vitro it is common for appreciable subpopulations of proteins to remain misfolded, soluble, and evade the refolding action of chaperones for very long times. Further, these results suggest that this happens because these misfolded subpopulations are near-native and therefore interact with chaperones to a similar extent as properly folded proteins. More broadly, these results indicate a mechanism in which long-time scale changes in protein structure and function can persist in cells because some protein’s non-native states can bypass components of the proteostasis machinery.TEASERNear-native, misfolded protein conformations explain why some soluble proteins fail to refold in the presence of chaperones.

scholarly journals Multiple prebiotic metals mediate translation

2018 ◽  
Author(s):  
Marcus S. Bray ◽  
Timothy K. Lenz ◽  
Jay William Haynes ◽  
Jessica C. Bowman ◽  
Anton S. Petrov ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Tertiary Structure ◽  
Cell Function ◽  
Living Organism ◽  
Translation System ◽  
Ancestral State ◽  
Functional Protein ◽  
Functional Roles ◽  
And Function

ABSTRACTToday, Mg2+is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’HydroxylAcylation analyzed byPrimerExtension) was used to compare the effect of Mg2+, Fe2+, and Mn2+on the tertiary structure of rRNA. Then, we usedin vitrotranslation reactions to test whether Fe2+or Mn2+could mediate protein production, and quantified ribosomal metal content. We found that: (i) Mg2+, Fe2+, and Mn2+had strikingly similar effects on rRNA folding; (ii) Fe2+and Mn2+can replace Mg2+as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+and Mn2+were abundant, these findings suggest that Fe2+and Mn2+played a role in early ribosomal evolution.SIGNIFICANCERibosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O2, high Fe2+, high Mn2+). Our results expand the roles of Fe2+and Mn2+in ancient and extant biochemistry as cofactors for ribosomal structure and function.

Molecular-Scale Studies of Proteins at Model Biomaterial Surfaces by Atomic Force Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 124-125
Author(s):  
Christopher A. Siedlecki
Keyword(s):  
Atomic Force Microscopy ◽  
Tertiary Structure ◽  
Molecular Level ◽  
Biological Response ◽  
Initial Step ◽  
Protein Layer ◽  
Force Microscopy ◽  
Atomic Force ◽  
Biomaterial Surfaces ◽  
And Function

A widely accepted tenet of biomaterials research is that the initial step following contact of a synthetic material with blood is the rapid adsorption of plasma proteins. The composition of this adsorbed protein layer is dependent on a variety of factors, including the surface properties of the implant material and the nature of the adsorbing proteins, and the composition and function of this protein layer is important in the subsequent biological response and ultimately the success or failure of the implanted material. While a great amount of effort has gone into developing structure/function responses for implanted biomaterials, there is still much about the molecular level interactions to be determined. We utilized atomic force microscopy (AFM) to investigate the molecular-level interactions of proteins with model biomaterial substrates. The AFM is unique in that it offers the opportunity to characterize interfacial environments, determine material properties, measure protein/surface interaction forces, and visualize the tertiary structure of adsorbed proteins.

Functional ABCG2 Is Expressed on CML Stem Cells and Its Inhibition Selectively Depletes CML CD34+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 716-716
Author(s):  
Joanne C. Mountford ◽  
Diane Gilmour ◽  
Susan M. Graham ◽  
Niove E. Jordanides ◽  
Siobhan McMillan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Efflux Pump ◽  
Residual Disease ◽  
Chronic Phase ◽  
Cell Number ◽  
Similar Degree ◽  
Cd34 Cells ◽  
And Function

Abstract We have previously described a population of deeply, but reversibly, quiescent stem cells (qSC) found in patients with chronic phase (CP) CML at diagnosis. In vitro studies have proven this population to be highly insensitive to imatinib mesylate (IM; Gleevec, STI571) induced killing, and more worryingly shown that qSC are accumulated after CML CD34+ cells are treated with IM. As it is likely that CML qSC closely resemble normal HSC, we hypothesise that they too may express the stem cell-associated ABCG2 and have therefore examined the expression and function of this drug efflux pump on CML cells. In agreement with other studies we show the interaction between ABCG2 and IM. Using ABCG2 over-expressing cells (AML6.2 and HL60-BCRP) we found that ≥0.5μM IM reduced efflux of the ABCG2 substrate BODIPY-Prazosin by a similar degree as the inhibitor fumitremorgin C (FTC; 10μM). We have now examined expression and function of ABCG2 on primary CML cells taken from patients in chronic phase (CP) and prior to any treatment. Quantitative Taqman analysis of 8 CD34+ enriched (≥90%+) CML samples revealed that the level of expression is 2.46 fold higher than that in normal mobilised CD34+ cells (n=8 CML, n=4 normal). In addition, we undertook microarray analysis of normal or CML CP CD34+ cells fractionated according to cell cycle using Hoechst-Pyronin (G0, G1 and G2/S/M). These analyses (n=3 normal, n=5 CML) show that at all stages of the cycle CML cells express more ABCG2 than normal cells and that G0 CML cells express 2.48 fold more than those in G1 , confirming both the over-expression in CML and relationship to the most primitive subset of cells. Using the antibody BXP21 we found that 8 of 9 samples contain ABCG2+ve cells (5 of 9 ≥60% of cells ABCG2+). We also examined the function of ABCG2 on CML CD34+ cells by performing efflux assays, 4 of 6 showed efflux that was inhibited by 10μM FTC or ≥0.5μM IM, and this efflux capacity correlated with BXP21 staining. We therefore considered whether the combination of IM therapy and ABCG2 inhibition would overcome the accumulation of CML qSCs we have previously reported after treatment with IM. Using CFSE to track cell division we treated CD34+ enriched CML samples with 5μM IM +/− FTC or with 10μM FTC alone for 3 days. In comparison to untreated controls 5μM IM reduced the total number of cells to 31.9±9.2 % and the number of CD34+ cells to 43.2±17.6%. However, the non-cycling qSC significantly increased to 318±75.8% of control. In contrast, the ABCG2 inhibitor FTC did not effect a reduction in total cells (99.5±11.9%) but gave a significant reduction of CD34+ cells (58.6±8.4%; p=0.02) and no accumulation of qSC (104.6±33.8%) when used alone. We saw no cumulative effect when IM and FTC were given concurrently. These data suggest strongly that FTC may be used to deplete CD34+ ‘stem cells’ from CML, as the total cell number is unchanged it is likely that this depletion is by the induction of differentiation. We propose that the expression of ABCG2 may be clinically significant in CP CML and that inhibition of this pump may result in a ‘stem cell targeted therapy’ that could be followed by IM treatment to reduce the tumor load. Such reduction of CML stem cells would result in elimination of minimal residual disease and effect a lasting remission.

scholarly journals Htm1p–Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

2016 ◽  
Vol 113 (28) ◽  
pp. E4015-E4024 ◽  
Author(s):  
Yi-Chang Liu ◽  
Danica Galonić Fujimori ◽  
Jonathan S. Weissman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Protein Disulfide Isomerase ◽  
Misfolded Proteins ◽  
Disulfide Isomerase ◽  
Licensing Factor ◽  
Folded Proteins ◽  
Protein Disulfide

Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p–Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae. We reconstitute this step in vitro with Htm1p–Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p–Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p–Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

scholarly journals Response differences of HepG2 and Primary Mouse Hepatocytes to morphological changes in electrospun PCL scaffolds

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thomas S. R. Bate ◽  
Victoria L. Gadd ◽  
Stuart J. Forbes ◽  
Anthony Callanan
Keyword(s):  
Cell Attachment ◽  
Morphological Changes ◽  
Functional Markers ◽  
Functional Protein ◽  
Stage Disease ◽  
Primary Mouse ◽  
Drug Treatments ◽  
End Stage ◽  
And Function

AbstractLiver disease cases are rapidly expanding across the globe and the only effective cure for end-stage disease is a transplant. Transplant procedures are costly and current supply of donor livers does not satisfy demand. Potential drug treatments and regenerative therapies that are being developed to tackle these pressing issues require effective in-vitro culture platforms. Electrospun scaffolds provide bio-mimetic structures upon which cells are cultured to regulate function in-vitro. This study aims to shed light on the effects of electrospun PCL morphology on the culture of an immortalised hepatic cell line and mouse primary hepatocytes. Each cell type was cultured on large 4–5 µm fibres and small 1–2 µm fibres with random, aligned and highly porous cryogenically spun configurations. Cell attachment, proliferation, morphology and functional protein and gene expression was analysed. Results show that fibre morphology has a measurable influence on cellular morphology and function, with the alteration of key functional markers such as CYP1A2 expression.

Screening Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry

2021 ◽  
Author(s):  
Florian Busch ◽  
Zachary VanAernum ◽  
Stella M. Lai ◽  
Venkat Gopalan ◽  
Vicki Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Tertiary Structure ◽  
Direct Analysis ◽  
Native Mass Spectrometry ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Functional Protein ◽  
Translation Rate

Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express robustly in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to screen<br>multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography–mass spectrometry-based method for rapid, direct analysis of target proteins in cell lysates. This online approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based buffer exchange (OBE) and native mass spectrometry (nMS). The use of IMAC-OBE-nMS to optimize conditions for large-scale protein production should expedite structural biology and biotherapeutic initiatives.<br>

scholarly journals Multiple prebiotic metals mediate translation

2018 ◽  
Vol 115 (48) ◽  
pp. 12164-12169 ◽  
Author(s):  
Marcus S. Bray ◽  
Timothy K. Lenz ◽  
Jay William Haynes ◽  
Jessica C. Bowman ◽  
Anton S. Petrov ◽  
...  
Keyword(s):  
Metal Content ◽  
Protein Production ◽  
Tertiary Structure ◽  
In Vitro Translation ◽  
Translation System ◽  
Ancestral State ◽  
Functional Protein ◽  
Functional Roles ◽  
Shape Selective

Today, Mg2+is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, and high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) was used to compare the effect of Mg2+, Fe2+, and Mn2+on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+or Mn2+could mediate protein production, and quantified ribosomal metal content. We found that (i) Mg2+, Fe2+, and Mn2+had strikingly similar effects on rRNA folding; (ii) Fe2+and Mn2+can replace Mg2+as the dominant divalent cation during translation of mRNA to functional protein; and (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+and Mn2+were abundant, these findings suggest that Fe2+and Mn2+played a role in early ribosomal evolution.

scholarly journals An unexpected effect of TNF-α on F508del-CFTR maturation and function

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 218 ◽  
Author(s):  
Sara Bitam ◽  
Iwona Pranke ◽  
Monika Hollenhorst ◽  
Nathalie Servel ◽  
Christelle Moquereau ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Fluid Transport ◽  
Inflammatory Responses ◽  
Underlying Mechanism ◽  
Transmembrane Conductance Regulator ◽  
Functional Protein ◽  
Chloride Currents ◽  
Tnf Α ◽  
Folded Proteins ◽  
And Function

Cystic fibrosis (CF) is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT) CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml) of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE) leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC) signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular.

scholarly journals Mg2+ binding triggers rearrangement of the IM30 ring structure, resulting in augmented exposure of hydrophobic surfaces competent for membrane binding

2018 ◽  
Vol 293 (21) ◽  
pp. 8230-8241 ◽  
Author(s):  
Jennifer Heidrich ◽  
Benedikt Junglas ◽  
Natalia Grytsyk ◽  
Nadja Hellmann ◽  
Kristiane Rusitzka ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding ◽  
Hydrophobic Surface ◽  
Ring Structure ◽  
Membrane Fusion Protein ◽  
Double Ring ◽  
Quaternary Structures ◽  
And Function

The “inner membrane–associated protein of 30 kDa” (IM30), also known as “vesicle-inducing protein in plastids 1” (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+. However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of ∼1 mm. Mg2+ binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation in vitro because of increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.

scholarly journals Htm1p-Pdi1p is a folding sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

2016 ◽  
Author(s):  
Yi-Chang Liu ◽  
Danica Galonić Fujimori ◽  
Jonathan S. Weissman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Misfolded Proteins ◽  
Licensing Factor ◽  
Folded Proteins

AbstractOur understanding of how the endoplasmic reticulum-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the Htm1p-Pdi1p complex acts as a folding-sensitive mannosidase for catalyzing this first committed step. We reconstitute this step in vitro with Htm1p-Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p-Pdi1p is a glycoprotein-specific mannosidase, which preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p-Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

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