Proper Folding Research Articles

Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability

Hevin is a secreted extracellular matrix protein that is encoded by the SPARCL1 gene. Recent studies have shown that Hevin plays an important role in regulating synaptogenesis and synaptic plasticity. Mutations in the SPARCL1 gene increase the risk of autism spectrum disorder (ASD). However, the molecular basis of how mutations in SPARCL1 increase the risk of ASD is not been fully understood. In this study, we show that one of the SPARCL1 mutations associated with ASD impairs normal Hevin secretion. We identified Hevin mutants lacking the EF-hand motif through analyzing ASD-related mice with vulnerable spliceosome functions. Hevin deletion mutants accumulate in the endoplasmic reticulum (ER), leading to the activation of unfolded protein responses. We also found that a single amino acid substitution of Trp647 with Arg in the EF-hand motif associated with a familial case of ASD causes a similar phenotype in the EF-hand deletion mutant. Importantly, molecular dynamics (MD) simulation revealed that this single amino acid substitution triggers exposure of a hydrophobic amino acid to the surface, increasing the binding of Hevin with molecular chaperons, BIP. Taken together, these data suggest that the integrity of the EF-hand motif in Hevin is crucial for proper folding and that ASD-related mutations impair the export of Hevin from the ER. Our data provide a novel mechanism linking a point mutation in the SPARCL1 gene to the molecular and cellular characteristics involved in ASD.

PETase from Ideonella sakaiensis: towards facile bacterial expression system

One of the most promising PET-hydrolysing enzyme, IsPETase, originates from 'plastic-consuming' bacteria Ideonella sakaiensis and operates at moderate temperatures (30-40 °C) in aqueous environment, showing far higher hydrolysing efficiency than several cutinases. Accordingly, the development of proper recombinant expression system providing facile access to the isolated/purified IsPETase is also of high interest. In our aim to produce active recombinant IsPETase, the unsuccessful expression registered by employing several reported expression systems, directed us towards the optimization/ development of a facile/accessible recombinant expression system suitable for IsPETase production. Testing several plasmid constructs, providing different N- or C-terminal affinity tags for the expression of full-length or truncated (residues 28-290) IsPETase and various E. coli expression hosts, revealed several non-reported issues hindering the recombinant expression of IsPETase. After optimization of the construct and expression host, the use of N-terminal His-tag and Rosetta-gami B expression host provided recombinant IsPETase in high purity and titer-yield. The thermal denaturation profile and PET-hydrolysing activity of the obtained IsPETase agree with the reported data, supporting proper folding of the purified protein. The results support that the in vivo folding process of IsPETase might be differently affected among the different E. coli host strains, moreover, underline the importance of the proper selection of the cloning strategy for the successful expression of the IsPETase.

Low protein expression enhances phenotypic evolvability by intensifying selection on folding stability.

Protein abundance affects the evolution of protein genotypes, but we do not know how it affects the evolution of protein phenotypes. Here we investigate the role of protein abundance in the evolvability of green fluorescent protein (GFP) towards the novel phenotype of cyan fluorescence. We evolve GFP in E. coli through multiple cycles of mutation and selection and show that low GFP expression facilitates the evolution of cyan fluorescence. A computational model whose predictions we test experimentally helps explain why: lowly expressed proteins are under stronger selection for proper folding, which facilitates their evolvability on short evolutionary time scales. The reason is that high fluorescence can be achieved by either few proteins that fold well or by many proteins that fold less well. In other words, we observe a synergy between a protein's scarcity and its stability. Because many proteins meet the essential requirements for this scarcity-stability synergy, it may be a widespread mechanism by which low expression helps proteins evolve new phenotypes and functions.

RapD Is a Multimeric Calcium-Binding Protein That Interacts With the Rhizobium leguminosarum Biofilm Exopolysaccharide, Influencing the Polymer Lengths

Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix’s properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.

S153: DECRYPTING THE ROLE OF HSP90Α AND Β ISOFORMS TO OVERCOME RESISTANCE IN BCR-ABL1 LEUKEMIA

Background: The BCR-ABL1 fusion protein, which results from the chromosomal translocation t(9;22)(q34;q11) is detected in over 95% of CML, 25% of adult BCP-ALL and 3-5% of childhood BCP-ALL patients. The development of tyrosine kinase inhibitors (TKi) has enabled targeted treatment, however development of resistance is common and the BCR-ABL1 leukemia subtype is still associated with poor prognosis. Among the chaperone proteins involved in maintaining the homeostasis of cancerous cells, heat shock protein 90 (HSP90) have been widely studied, due to its crucial involvement in stabilizing a variety of oncogenic proteins, including BCR-ABL1 and STAT3/5. Therefore, inhibition of HSP90 can be an effective alternative approach to target TKi resistant and BCR-ABL1T315I mutant expressing cells. In the past, over 17 HSP90 inhibitors (HSP90i) have been evaluated in clinical trials, however associated resistances (e.g. HSR) and dose limited toxicity have thus far precluded their clinical approval. Aims: In mammalian cells, there are two predominant cytosolic isoforms of HSP90, a stress-inducible HSP90α and a constitutively expressed HSP90β isoform. In the past, the distinct roles of the isoforms were studied by applying pan- and isoform-targeted HSP90i. We aimed to decrypt the role of cytosolic HSP90 isoforms using genetically edited models and to identify novel therapeutic vulnerabilities to target therapy refractory BCR-ABL1+ leukemia. Methods: To study the kinetic implications of the resistance mechanism associated with pharmacological targeting of HSP90, we generated a transient knockdown (Kd) and a stable CRISPR-Cas9 based knockout (KO) model of HSP90α/β in BCR-ABL1+ cells. The edited cells were analyzed in ex vivo functional assays (e.g. IF staining and WB) and their transplantation efficiency was determined in NSG mice. Global transcriptomic and proteomic profiling was performed in conjunction with high throughput drug screening (HTDS) to identify therapeutic vulnerabilities upon loss of HSP90α/β. Results: HSP90α/β-KO or -Kd did not affect the expression of other HSP90 paralogues (TRAP1 and GRP94). Only HSP90β-KO cells displayed hyperactive pro-survival HSR. Other reported HSP90 isoform-dependent clients, such as Survivin, CDK4 and CDK6 were also not affected upon KO or Kd of HSP90α/β. As HSP90 is involved in stabilizing, proper folding and subcellular localization of BCR-ABL1 protein, immunofluorescence imaging demonstrated two fold higher abundance of BCR-ABL1 foci (cytoplasmic/nucleocytoplasmic region) in HSP90α KO cells, with hyperactive BCR-ABL1 and downstream signaling (pBCR-ABL and pSTAT5a). When KO cells were transplanted into NSG mice (n=5 mice per arm) the engraftment of the HSP90α-KO cells was significantly reduced with a prolonged overall survival of the animals (19 days, p=0.0083) as compared to HSP90β-KO and control group. To find possible target (s) in order to circumvent resistance associated with the use HSP90i, we performed HTDS with HSP90α/β KO cells, which revealed distinct sensitivities toward certain classes of inhibitors. One of these classes was CDK7i, which was differentially active in HSP90α KO cells. These results can be explained by our global transcriptomic results, which revealed hyperactive androgen receptor signaling in HSP90α KO cells (GSEA), which is known to be a prognostic marker for CDK7 inhibition. Summary/Conclusion: In vivo targeting of HSP90α is a promising approach to target BCR-ABL1+ leukemia cells. Moreover, the combination with CDK7i may help to overcome the resistance associated with clinical use of pan HSP90i.

Abstract 3993: ERO1α as a potential target in EGFR driven non-small cell lung cancer

Abstract Endoplasmic Reticulum Oxidoreductase-1 alpha (ERO1α) is an endoplasmic reticulum (ER) resident protein responsible for oxidation of Protein Disulfide Isomerase (PDI). The molecular interactions between ERO1α and PDI allow for proper protein folding to occur. Recent reports indicate that ERO1α expression is a poor prognostic indicator of survival in multiple cancer types including multiple myeloma, lung cancer, esophageal carcinoma, hepatocellular carcinoma, and diffuse large B-cell lymphoma. In this study we utilized genetic strategies to determine the role of ERO1α in EGFR driven Non-Small Cell Lung Cancer (NSCLC). Our data indicate that CRISPR knockout of ERO1α leads to decreased clonogenicity in both PC-9 and HCC4006 cell lines. The phenotype can be partially rescued upon addition of conditioned media derived from the control cell lines. The data suggest that ERO1α expression is required for correct protein folding of a secreted factor that contributes to clonogenicity and growth. PC-9 ERO1α knockout cells injected into SCID-Beige animals led to increased overall survival (OS) compared to animals injected with PC-9 control cells. (PC-9 Control OS = 100 days, PC-9 ERO1α Knockout OS =154 days p=0.0045 **). Interestingly, ERO1α knockout cell lines still maintained sensitivity to standard of care agent Osimertinib. We identified T151742 as an ERO1α inhibitor that was approximately 2-fold more potent compared to the previously published ERO1α inhibitor EN460. A cellular thermal shift assay was performed to determine specificity of T151742 toward ERO1α compared to other FAD containing enzymes. We show that T151742 had a higher specificity towards ERO1α in comparison to other flavoenzymes such as ERO1β and LSD-1. Finally, our data indicate that PC-9 ERO1α knockout cell lines were ~2-fold resistant to T151742 by MTT but were completely resistant when using a 3D cell culture model suggesting T151742 is reliant on ERO1α in a 3D system. Finally, treatment of PC9 and HCC4006 cells with T151742 in combination with Osimertinib was synergistic in vitro. Taken together, this suggests ERO1α may be a viable target for EGFR driven NSCLC and that future studies will elucidate the use of T157142 in vivo as a single agent and in combination with EGFR inhibitors using in vivo studies. Citation Format: Brennan D. Johnson, Wei-Chih Chen, Werner J. Geldenhuys, Lori A. Hazlehurst. ERO1α as a potential target in EGFR driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3993.

Abstract 5851: Negative regulation of p53 by ZnT1

Abstract Rationale and Background: p53 is a powerful tumor suppressor protein that also autorepresses by inducing expression of its own negative regulators, such as MDM2. Here we explore another potential mechanism for p53 autorepression, regulation of zinc metabolism. Proper folding of p53 requires zinc binding and cancer-associated mutations in p53 often lower the affinity of the mutant proteins for zinc. Our previous work showed that ZMC1, a zinc metallochaperone, can reactivate certain p53 mutants by raising intracellular zinc concentrations. Here we show that p53 activates transcription of ZnT1. ZnT1 can then export zinc from the cell, which in turn lowers p53 activity. Methods: To explore the relationship between ZnT1 and p53, we studied several human cancer cell lines under various conditions, including ZnT1 overexpression, ZnT1 silencing, and p53 activation. For silencing gene expression, we used CRISPR-Cas9 with a lentiviral vector to knock out ZnT1 and also employed shRNAs to knockdown expression of p53 or ZnT1. To analyze the relationship between TP53 and ZnT1 genes in clinical samples, data from cBioPortal were examined. Results: ZnT1 transcription increased after p53 was activated in several p53+ cell lines but did not increase in isogenic p53-null cell lines, suggesting that ZnT1 transcription is dependent on p53. p53 also bound to the ZnT1 promoter by chromatin immunoprecipitation assay, and activated transcription of a ZnT1/luciferase promoter fusion. Overexpression of ZnT1 decreased p21 transcription, indicating lowered p53 function; conversely, knockdown of ZnT1 increased p21 transcription. By cBioPortal analysis, ZnT1 gene amplifications were mutually exclusive with TP53 gene mutations in breast and lung tumors. Conclusion: These data suggest that autorepression of p53 occurs in part due to lowered intracellular zinc caused by upregulation of ZnT1. Citation Format: Ben Brik, Kathleen Carino, Wade Narrow, Chris R. Harris, Xin Yu, Darren R. Carpizo. Negative regulation of p53 by ZnT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5851.

METTL18-mediated histidine methylation of RPL3 modulates translation elongation for proteostasis maintenance.

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a methylation substrate. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood to date. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traversal on Tyr codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the τ-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation reactions. Indeed, genome-wide ribosome profiling and an in vitro translation assay revealed that translation elongation at Tyr codons was suppressed by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a ribosome modification that ensures proteome integrity in cells.

Reversion of MRAP2 Protein Sequence Generates a Functional Novel Pharmacological Modulator for MC4R Signaling.

Simple SummaryReversion of the wild-type protein sequences of single transmembrane melanocortin accessory protein families (MRAP2) in mice and zebrafish created novel functional pharmacological modulators for regulating melanocortin 4 receptor (MC4R) signaling. All of the brand new reversed MRAP2 (rMRAP2) proteins could form proper dimeric topology on the plasma membrane and interact with and affect the ligand-stimulated pharmacological profiles of zebrafish and mouse MC4R signaling in vitro.As a member of the melanocortin receptor family, melanocortin 4 receptor (MC4R) plays a critical role in regulating energy homeostasis and feeding behavior, and has been proven as a promising therapeutic target for treating severe obesity syndrome. Numerous studies have demonstrated that central MC4R signaling is significantly affected by melanocortin receptor accessory protein 2 (MRAP2) in humans, mice and zebrafish. MRAP2 proteins exist as parallel or antiparallel dimers on the plasma membrane, but the structural insight of dual orientations with the pharmacological profiles has not yet been fully studied. Investigation and optimization of the conformational topology of MRAP2 are critical for the development of transmembrane allosteric modulators to treat MC4R-associated disorders. In this study, we synthesized a brand new single transmembrane protein by reversing wild-type mouse and zebrafish MRAP2 sequences and examined their dimerization, interaction and pharmacological activities on mouse and zebrafish MC4R signaling. We showed that the reversed zebrafish MRAPa exhibited an opposite function on modulating zMC4R signaling and the reversed mouse MRAP2 lost the capability for regulating MC4R trafficking but exhibited a novel function for cAMP cascades, despite proper expression and folding. Taken together, our results provided new biochemical insights on the oligomeric states and membrane orientations of MRAP2 proteins, as well as its pharmacological assistance for modulating MC4R signaling.

Mortalin/Hspa9 involvement and therapeutic perspective in Parkinson's disease.

By controlling the proper folding of proteins imported into mitochondria and ensuring crosstalk between the reticulum and mitochondria to modulate intracellular calcium fluxes, Mortalin is a chaperone protein that plays crucial roles in neuronal homeostasis and activity. However, its expression and stability are strongly modified in response to cellular stresses, in particular upon altered oxidative conditions during neurodegeneration. Here, we report and discuss the abundant literature that has highlighted its contribution to the pathophysiology of Parkinson’s disease, as well as its therapeutic and prognostic potential in this still incurable pathology.

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

245-LB: An Ankyrin-B–Mediated Protein Chaperone Mechanism Modulates Pancreatic ß-Cell Susceptibility to Proteotoxicity-Induced ER Stress and Apoptosis

Impaired insulin secretion contributes to the development and progression of diabetes. β-cells initially cope with the genetic, environmental, and diabetogenic stresses that diminish insulin secretion or the peripheral response to it by increasing insulin production. However, sustained increases in the insulin precursor proinsulin (PI) place additional demand on the secretory and protein quality control (PQC) machineries of β-cells, triggering endoplasmic reticulum (ER) and oxidative stress, mitochondrial dysfunction, and β-cell death. Thus, understanding the mechanisms that regulate β-cell responses to increased insulin secretory requirements is critical to prevent β-cell failure. We reported that the cytoskeletal scaffolding protein ankyrin-B (AnkB) regulates insulin secretion by maintaining normal levels of the inositol-1,4,5-triphosphate receptor (IP3R) at the ER and intracellular calcium in β-cells. IP3R levels and insulin secretion were impaired in knock-in mice carrying the p.R1788W AnkB (AnkB-RW) variant associated with type 2 diabetes (T2D) in Caucasians and Hispanics. Here, we show that selective loss of AnkB or expression of the T2D AnkB-RW variant lead to reduced insulin secretion and accumulation of PI and islet specific amyloid polypeptide in β-cells. These changes are accompanied by enhanced induction of the maladaptive unfolded protein response and apoptosis. Interestingly, the T2D AnkB-RW variant lies within the binding site for the protein chaperone Dnajb1 (Hsp40) . Our results indicate that the Dnajb1-Hspa1a complex, which recognizes and delivers protein clients to Hsp70 chaperones to promote proper folding, refolding, or degradation, is downregulated in β-cells from β-cell-specific AnkB knockout or AnkB-RW mice. Increased proteotoxicity and reduced insulin secretion were also observed upon knockdown of Dnajb1 in rat INS1 832/13 cells. Together, our findings suggest that AnkB and its integration into the Dnajb1-Hspa1a pathway modulates PQC and promotes β-cell survival under diabetogenic conditions. Disclosure J. Tzeng: None. H. Hohmeier: None. D. Lorenzo: None.

Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation.

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

Regulation of Translation, Translocation, and Degradation of Proteins at the Membrane of the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) of mammalian cells is the central organelle for the maturation and folding of transmembrane proteins and for proteins destined to be secreted into the extracellular space. The proper folding of target proteins is achieved and supervised by a complex endogenous chaperone machinery. BiP, a member of the Hsp70 protein family, is the central chaperone in the ER. The chaperoning activity of BiP is assisted by ER-resident DnaJ (ERdj) proteins due to their ability to stimulate the low, intrinsic ATPase activity of BiP. Besides their co-chaperoning activity, ERdj proteins also regulate and tightly control the translation, translocation, and degradation of proteins. Disturbances in the luminal homeostasis result in the accumulation of unfolded proteins, thereby eliciting a stress response, the so-called unfolded protein response (UPR). Accumulated proteins are either deleterious due to the functional loss of the respective protein and/or due to their deposition as intra- or extracellular protein aggregates. A variety of metabolic diseases are known to date, which are associated with the dysfunction of components of the chaperone machinery. In this review, we will delineate the impact of ERdj proteins in controlling protein synthesis and translocation under physiological and under stress conditions. A second aspect of this review is dedicated to the role of ERdj proteins in the ER-associated degradation pathway, by which unfolded or misfolded proteins are discharged from the ER. We will refer to some of the most prominent diseases known to be based on the dysfunction of ERdj proteins.

The nonstructural protein 2C of Coxsackie B virus has RNA helicase and chaperoning activities.

RNA-remodeling proteins, including RNA helicases and chaperones, play vital roles in the remodeling of structured RNAs. During viral replication, viruses require RNA-remodeling proteins to facilitate proper folding and/or re-folding the viral RNA elements. Coxsackieviruses B3 (CVB3) and Coxsackieviruses B5 (CVB5), belonging to the genus Enterovirus in the family Picornaviridae, have been reported to cause various infectious diseases such as hand-foot-and-mouth disease, aseptic meningitis, and viral myocarditis. However, little is known about whether CVB3 and CVB5 encode any RNA remodeling proteins. In this study, we showed that 2C proteins of CVB3 and CVB5 contained the conserved SF3 helicase A, B, and C motifs, and functioned not only as RNA helicase that unwound RNA helix bidirectionally in an NTP-dependent manner, but also as RNA chaperone that remodeled structured RNAs and facilitated RNA strand annealing independently of NTP. In addition, we determined that the NTPase activity and RNA helicase activity of 2C proteins of CVB3 and CVB5 were dependent on the presence of divalent metallic ions. Our findings demonstrate that 2C proteins of CVBs possess RNA-remodeling activity and underline the functional importance of 2C protein in the life cycle of CVBs.

The structure of Deinococcus radiodurans transcriptional regulator HucR retold with the urate bound

HucR is a MarR family protein of Deinococcus radiodurans, which binds tightly to the intergenic region of HucR and the uricase gene to inhibit their expression. Urate (or uric acid) antagonizes the repressor function of HucR by binding to HucR to impede its association with the cognate DNA. The previously reported crystal structure of HucR was without the bound urate showing significant structural homology to other MarR structures. In this paper, we report the crystal structure of HucR determined with the urate bound. However, despite the fact that the urate is found at a site well-known to harbor ligands in other MarR family proteins, the overall HucR structure indicates that no significant change in structure takes place with the urate bound. Structure analysis further suggests that the urate interaction in HucR is mediated by histidine/glutamate side chains and ordered water molecules stabilized by various residues. Such interaction is quite unique compared to other known structural interactions between urate and its binding proteins. Furthermore, structural comparison of the apo- and the urate bound forms allows us to hypothesize that the Trp20-mediated water network in the apo-form stabilizes the proper HucR fold for cognate DNA binding, and that urate binding, also via Trp20, and the consequent reorganization of water molecules in the binding pocket, likely disrupts the DNA binding configuration to result in the attenuated DNA binding.

Investigating the Determinants of Substrate Binding Orientation within the E. coli Hsp70 Chaperone Protein

Molecular chaperone proteins play a critical role in maintenance of the cell’s proteome through protein quality control. Heat shock protein 70 chaperones (Hsp70s) are an evolutionarily conserved and diverse family of chaperone proteins that assist with proper protein folding and prevention of aggregation in the cell. Previous research has shown that the substrate‐binding domain of the E. coli Hsp70, DnaK, contains binding pockets of low stringency extending bilaterally from a highly hydrophobic central pocket. The SBD interacts with short hydrophobic sequences of substrates by “reading” the sequence. The SBD will then bind the substrate in one of two orientations: an N‐C, or forward, orientation, or a C‐N, reverse, orientation. Research done thus far on substrate binding has not revealed an exact mechanism that determines orientation. In this study, peptides possessing the same sequence with opposing backbone directions were used to test the significance of backbone interactions with the SBD as well as residue side chains and their interactions with the SBD. The analysis of the bound orientation of these peptides is accomplished by disulfide cross‐linking using peptides containing an N‐terminal Cys and SBD proteins with a Cys introduced in such a way where it can be reached in only one orientation. The results indicated slight energetic favorable biases for the interactions of residue side chains and the SBD pockets. The results also place a high importance on leucine residues in substrates as a determinant of optimal pocket ‐ residue fit and binding orientation. Altogether, the data suggest that binding occurs in the orientation that supports the most optimal fit of residue side chains into binding pockets, which reveals further intricacies and selectivity involved in substrate binding for Hsp70s.

Salty Enzymes: Protein Splicing Dependence on Salt Concentrations

Halophiles are organisms that thrive in very high salt concentrations. Enzymes from halophiles tend to have a negative surface charge and a low isoelectric point to allow for stable folding. We are interested in converting a halophilic enzyme that requires high salt for activity and proper folding into one that is functional at more moderate salt concentrations. Our model enzyme is DNA polymerase II from Halobacterium salinarum, which contains an intein. Inteins are intervening peptide sequences within proteins that are spliced out from their flanking polypeptides, or exteins, which then ligate to form a functional protein. We have induced mutations to the intein that facilitate a splicing reaction at lower salt concentrations. Changing negatively charged amino acids on the surface of the intein to corresponding neutral or positive charged residues observed in homologous inteins in non‐halophilic extremophiles induces the splicing reaction at lower salt concentrations, especially when coupled with a change of the C‐terminal Gln to the more highly conserved Asn. As few as two residue changes can dramatically alter the salt‐dependence of the activity, showing that subtle changes to surface residues can result in conditional folding and activity.

The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS‐CoV‐2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin‐converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post‐translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS‐1 cells in the presence or absence of N‐butyldeoxynojirimycin (NB‐DNJ), an inhibitor of the ER‐located a‐glucosidases I and II, 1‐deoxymannojirimycin (dMM), an inhibitor of Golgi‐mannosidase I, or benzyl‐N‐acetyl‐α‐galactosaminide (benzyl‐GalNac), an inhibitor of O‐glycosylation. The data demonstrate that NB‐DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl‐GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB‐DNJ, as well as O‐glycans that are modulated by benzyl‐GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu‐3 and intestinal Caco‐2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB‐DNJ and dMM, while this interaction was enhanced in Calu‐3 cells upon inhibition of O‐glycosylation by benzyl‐GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB‐DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.

Myosin chaperone, UNC‐45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The intestinal epithelium creates a protective barrier separating the luminal content and the immune system in the gut. Leakiness of the intestinal epithelial barrier and inefficient healing of mucosal wounds represent common manifestations of inflammatory bowel diseases (IBD). Integrity of the gut barrier is mediated by different adhesive structures, including adherens junctions (AJ), tight junctions (TJ) and focal adhesion (FA). Functions of these adhesive structures depend on their association with the cortical actin cytoskeleton and particularly, to a major actin motor protein, non‐muscle myosin II (NM‐II). While biochemical mechanisms that regulate activity of NM‐II in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. In this study, we focused on UNC‐45A, a cytoskeletal chaperone that is essential for proper folding of NM‐II heavy chains and myofilament assembly. We found abundant expression of UNC‐45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC‐45A were decreased in colonic epithelium of patients with ulcerative colitis in parallel to diminished expression of NM‐IIA and NM‐IIC isoforms. CRISPR/Cas9‐mediated knock‐out of UNC‐45A in HT‐29 and SK‐CO15 IEC disrupted epithelial barrier integrity, impaired AJ and TJ assembly, and cell migration. Consistently, decreased UNC‐45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti‐migratory effects of UNC‐45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC‐45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC‐45A in controlling IEC barrier function, junctional assembly and migration. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.