Substrate-binding Domain Research Articles

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Conformational and oligomeric states of SPOP from small-angle X-ray scattering and molecular dynamics simulations.

Speckle-type POZ protein (SPOP) is a substrate adaptor in the ubiquitin proteasome system, and plays important roles in cell-cycle control, development, and cancer pathogenesis. SPOP forms linear higher-order oligomers following an isodesmic self-association model. Oligomerization is essential for SPOP's multivalent interactions with substrates, which facilitate phase separation and localization to biomolecular condensates. Structural characterization of SPOP in its oligomeric state and in solution is, however, challenging due to the inherent conformational and compositional heterogeneity of the oligomeric species. Here, we develop an approach to simultaneously and self-consistently characterize the conformational ensemble and the distribution of oligomeric states of SPOP by combining small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations. We build initial conformational ensembles of SPOP oligomers using coarse-grained molecular dynamics simulations, and use a Bayesian/maximum entropy approach to refine the ensembles, along with the distribution of oligomeric states, against a concentration series of SAXS experiments. Our results suggest that SPOP oligomers behave as rigid, helical structures in solution, and that a flexible linker region allows SPOP's substrate-binding domains to extend away from the core of the oligomers. Additionally, our results are in good agreement with previous characterization of the isodesmic self-association of SPOP. In the future, the approach presented here can be extended to other systems to simultaneously characterize structural heterogeneity and self-assembly.

P4HA2 induces hepatic ductular reaction and biliary fibrosis in chronic cholestatic liver diseases.

Prolyl-4-hydroxylases (P4Hs) are key enzymes in collagen synthesis. The P4HA subunit (P4HA1, P4HA2, and P4HA3) contains a substrate binding and catalyzation domain. We postulated that P4HA2 would play a key role in the cholangiocyte pathology of cholestatic liver diseases. We studied humans with primary biliary cholangitis (PBC) and Primary sclerosing cholangitis (PSC), P4HA2 -/- mice injured by DDC, and P4HA2 -/- /MDR2 -/- double knockout mice. A parallel study was performed in patients with PBC, PSC, and controls using immunohistochemistry and immunofluorescence. In the murine model, the level of ductular reaction and biliary fibrosis were monitored by histology, qPCR, immunohistochemistry, and Western blotting. Expression of Yes1 Associated Transcriptional Regulator (YAP) phosphorylation was measured in isolated mouse cholangiocytes. The mechanism of P4HA2 was explored in RBE and 293T cell lines by using qPCR, Western blot, immunofluorescence, and co-immunoprecipitation. The hepatic expression level of P4HA2 was highly elevated in patients with PBC or PSC. Ductular reactive cholangiocytes predominantly expressed P4HA2. Cholestatic patients with more severe liver injury correlated with levels of P4HA2 in the liver. In P4HA2 -/- mice, there was a significantly reduced level of ductular reaction and fibrosis compared with controls in the DDC-induced chronic cholestasis. Decreased liver fibrosis and ductular reaction were observed in P4HA2 -/- /MDR2 -/- mice compared with MDR2 -/- mice. Cholangiocytes isolated from P4HA2 -/- /MDR2 -/- mice displayed a higher level of YAP phosphorylation, resulting in cholangiocytes proliferation inhibition. In vitro studies showed that P4HA2 promotes RBE cell proliferation by inducing SAV1 degradation, eventually resulting in the activation of YAP. P4HA2 promotes hepatic ductular reaction and biliary fibrosis by regulating the SAV1-mediated Hippo signaling pathway. P4HA2 is a potential therapeutic target for PBC and PSC.

Dissecting the Enantioselective Neurotoxicity of Isocarbophos: Chiral Insight from Cellular, Molecular, and Computational Investigations.

Chiral organophosphorus pollutants are found abundantly in the environment, but the neurotoxicity risks of these asymmetric chemicals to human health have not been fully assessed. Using cellular, molecular, and computational toxicology methods, this story is to explore the static and dynamic toxic actions and its stereoselective differences of chiral isocarbophos toward SH-SY5Y nerve cells mediated by acetylcholinesterase (AChE) and further dissect the microscopic basis of enantioselective neurotoxicity. Cell-based assays indicate that chiral isocarbophos exhibits strong enantioselectivity in the inhibition of the survival rates of SH-SY5Y cells and the intracellular AChE activity, and the cytotoxicity of (S)-isocarbophos is significantly greater than that of (R)-isocarbophos. The inhibitory effects of isocarbophos enantiomers on the intracellular AChE activity are dose-dependent, and the half-maximal inhibitory concentrations (IC50) of (R)-/(S)-isocarbophos are 6.179/1.753 μM, respectively. Molecular experiments explain the results of cellular assays, namely, the stereoselective toxic actions of isocarbophos enantiomers on SH-SY5Y cells are stemmed from the differences in bioaffinities between isocarbophos enantiomers and neuronal AChE. In the meantime, the modes of neurotoxic actions display that the key amino acid residues formed strong noncovalent interactions are obviously different, which are related closely to the molecular structural rigidity of chiral isocarbophos and the conformational dynamics and flexibility of the substrate binding domain in neuronal AChE. Still, we observed that the stable "sandwich-type π-π stacking" fashioned between isocarbophos enantiomers and aromatic Trp-86 and Tyr-337 residues is crucial, which notably reduces the van der Waals' contribution (ΔGvdW) in the AChE-(S)-isocarbophos complexes and induces the disparities in free energies during the enantioselective neurotoxic conjugations and thus elucidating that (S)-isocarbophos mediated by synaptic AChE has a strong toxic effect on SH-SY5Y neuronal cells. Clearly, this effort can provide experimental insights for evaluating the neurotoxicity risks of human exposure to chiral organophosphates from macroscopic to microscopic levels.

Biochemical Characterization and Functional Analysis of Glucose Regulated Protein 78 from the Silkworm Bombyx mori.

The glucose regulated protein (GRP78) is an important chaperone for various environmental and physiological stimulations. Despite the importance of GRP78 in cell survival and tumor progression, the information regarding GRP78 in silkworm Bombyx mori L. is poorly explored. We previously identified that GRP78 expression was significantly upregulated in the silkworm Nd mutation proteome database. Herein, we characterized the GRP78 protein from silkworm B. mori (hereafter, BmGRP78). The identified BmGRP78 protein encoded a 658 amino acid residues protein with a predicted molecular weight of approximately 73 kDa and comprised of two structural domains, a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). BmGRP78 was ubiquitously expressed in all examined tissues and developmental stages by quantitative RT-PCR and Western blotting analysis. The purified recombinant BmGRP78 (rBmGRP78) exhibited ATPase activity and could inhibit the aggregating thermolabile model substrates. Heat-induction or Pb/Hg-exposure strongly stimulated the upregulation expression at the translation levels of BmGRP78 in BmN cells, whereas no significant change resulting from BmNPV infection was found. Additionally, heat, Pb, Hg, and BmNPV exposure resulted in the translocation of BmGRP78 into the nucleus. These results lay a foundation for the future identification of the molecular mechanisms related to GRP78 in silkworms.

E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In theself-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

SPOP is essential for DNA replication licensing through maintaining translation of CDT1 and CDC6 in HaCaT cells

Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition receptor for the cullin-3/RING ubiquitin E3 complex, leads to the ubiquitination of >40 of its target substrates. Since a variety of point mutations in the substrate-binding domain of SPOP have been identified in cancers, including prostate and endometrial cancers, the pathological roles of those cancer-associated SPOP mutants have been extensively elucidated. In this study, we evaluated the cellular functions of wild-type SPOP in non-cancerous human keratinocyte-derived HaCaT cells expressing wild-type SPOP gene. SPOP knockdown using siRNA in HaCaT cells dramatically reduced cell growth and arrested their cell cycles at G1/S phase. The expression of DNA replication licensing factors CDT1 and CDC6 in HaCaT cells drastically decreased on SPOP knockdown as their translation was inhibited. CDT1 and CDC6 downregulation induced p21 expression without p53 activation. Our results suggest that SPOP is essential for DNA replication licensing in non-cancerous keratinocyte HaCaT cells.

Protein-plastic interactions: The driving forces behind the high affinity of a carbohydrate-binding module for polyethylene terephthalate

Polyethylene terephthalate (PET) waste is a common pollutant in the environment, mainly due to resistance of the plastic to bio-degradation. Nevertheless, hydrolytic enzymes have been identified with activity on this substrate, which are continually being engineered to increase activity. Some insoluble biological polymers are degraded by enzymes with a multi-domain architecture, comprising of a catalytic domain, and a substrate-binding domain, such as a carbohydrate-binding module (CBM). Enzymes that degrade PET have been shown to have a higher activity when fused with these CBMs, indicating a promising route for engineering better enzymes for plastic bioprocessing. However, no detailed study of the affinity and binding mechanism of these domains on PET has yet been made. Here, we perform an in depth analysis of a binding domain from CBM family 2 on PET, showing that the affinity of the protein for the plastic is highly dependent on temperature and crystallinity of the plastic. We also investigate the mechanism of the interaction, and show how affinity may be engineered in both directions. CBM affinity for other synthetic polymers is also demonstrated for the first time. Our results demonstrate that the substrate affinity of fusion enzymes with binding modules can be tuned to the desired level.

Identification of key amino acid residues in OqxB mediated efflux of fluoroquinolones using site-directed mutagenesis

OqxB belongs to the RND (Resistance-Nodulation-Division) efflux pump family, recognized widely as a major contributor towards enhancing antimicrobial resistance. It is known to be predominantly present in all Klebsiella spp. and is attributed for its role in increasing resistance against an array of antibiotics like nitrofurantoin, quinolones, β-lactams and colistin. However, the presence of oqxB encoding this efflux pump is not limited only to Klebsiella spp., but is also found to occur via horizontal gene transfer in other bacterial genera like Escherichia coli, Enterobacter cloacae and Salmonella spp. Recently, we reported the crystal structure of OqxB and its structure-function relationship required for the efflux of fluoroquinolones. Extending these findings further, we characterized the structural architecture of this efflux pump along with identifying some critical amino acids at the substrate binding domain of OqxB. Based on our in silico modelling studies, both hydrophobic residues (F180, L280, L621, F626) and polar residues (R48, E50, E184, R157, R774) were found to be located at this site. The present work reports the importance of these key amino acid residues and the crucial ion–pair interactions at the substrate-binding pocket, thereby establishing their role in OqxB mediated efflux and the resultant resistance development against fluoroquinolones.

Molecular dynamics study of the mechanism of the client unfolded protein delivery from HSP40 to HSP70.

Heat Shock Protein (HSP) 70 is one of the molecular chaperones that supports protein folding. HSP70 is attracting attention as a target for drug discovery because of its involvement in cancer, neurodegenerative diseases, and so on. HSP70 consists of the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD), with a flexible-linker region between the NBD and SBD. The SBD has two regions, SBD-α, which contains alpha-helices, and SBD-β, which contains beta-sheets. In the HSP70 chaperone cycle, unfolded client proteins are delivered by a J-domain protein (JDP), such as a co-chaperone HSP40. The J-domain in the JDP binds to the ATP-bound HSP70 state when the substrate is delivered to HSP70. After that, the JDP stimulates the ATP hydrolysis of HSP70, and the conformational change to the ADP-bound HSP70 form. Then the HSP70 helps the client protein folding with the nucleotide exchange factor molecular cooperation. However, the complex structure of JDP and HSP70 has not been solved, and the mechanism of substrate transfer has not been in detail yet. In this research, to clarify the mechanism of the substrate transfer process from HSP40 to HSP70, we performed molecular dynamics simulations of HSP40, HSP70, and HSP70 with the J-domain. J-domain in the HSP40 has two forms, open and closed. We modeled the complex structure of JDP and HSP70 using the open form of the HSP40. We found that SBD-β of HSP70 locates between two J-domains of HSP40, and HSP40 fluctuates. Therefore, the SBD-β would scoop the substrate that binds to the HSP40. Also, we found that the J-domain binding induced the fluctuations around the ATP binding site and the substrate-binding site from the simulation of HSP70 and the J-domain complex structure.

S-27-7: ROLE OF THE UBIQUITIN LIGASE KLHL3 IN BLOOD PRESSURE AND ELECTROLYTE HOMEOSTASIS

Genetic studies on familial hypertensive and hypotensive disorders followed by the biochemical analysis have provided fundamental insights into the regulation of NaCl transport mechanisms in the renal nephron. One such example is the analysis of pseudohypoaldosteronism type II (also known as Gordon syndrome), which led to the identification of previously unrecognized constituents engaged in electrolyte and blood pressure homeostasis. Kelch-like 3 (KLHL3) is a component of cullin-RING (really interesting new gene) ubiquitin ligase whose mutations have been shown to cause pseudohypoaldosteronism type II through several mechanisms, including impaired ability to bind target substrates and altered protein stability. Studies to date have also provided insights into the physiological mechanisms that regulate KLHL3 function, which involves phosphorylation at the substrate-binding domain that is counter-regulated by protein kinase C and phosphatase calcineurin. Moreover, accumulating data indicate that these mechanisms are likely involved in the acquired forms of hypertension, particularly those associated with lifestyle related disorders such as obesity. Recent advance on the function of KLHL3 in the kidney, as well as its pathogenic roles in electrolyte disorders, will be reviewed and discussed.

Prediction of HCV E2 association with the host-cell chaperone, GRP78

BackgroundHepatitis C Virus (HCV) is the main causative factor for liver cirrhosis and for the development of liver cancer. E2 is an HCV structural protein responsible for virus entry to the host cell. GRP78 is the master regulator of the unfolded protein response mechanism in the Endoplasmic Reticulum (ER) in normal conditions. Under the stress of HCV infection or carcinogenesis, GRP78 is upregulated. Consequently, it escapes the ER retention and translocates to the cytoplasm and over the plasma membrane. AimThis study aims to predict the binding mode of HCV E2 to GRP78 protein. MethodsDue to the high sequence and structural conservation between the C554–C566 region of HCV E2 and the Pep42, cyclic peptide that is reported to target GRP78, we propose that this region of E2 can be the recognition site. We predict the possible binding mode between HCV E2 and GRP78 by implementing molecular docking and molecular dynamics simulation to test such proposed binding. ResultsThe simulations reveal a stable and highly potent (−111.2 docking score) binding of the HCV E2 C554–C566 peptide to GRP78 substrate-binding domain β (SBDβ). Moreover, the full-length HCV E2 also exhibits high binding affinity to GRP78 SBDβ (score = −107.5 ± 3.1), which is better than the association of GRP78 and Pep42. ConclusionsDefining the compulsory mode between HCV E2 and GRP78 is significant, so it would be possible to interfere with such binding to reduce the viral infection.

Effect of evolution of the C-terminal region on chaperone activity of Hsp70.

Dynamic interdomain interactions within the Hsp70 protein is the basis for the allosteric and functional properties of Hsp70s. While Hsp70s are generally conserved in terms of structure, allosteric behavior, and some overlapping functions, Hsp70s also contain variable sequence regions which are related to distinct functions. In the Hsp70 sequence, the part with the greatest sequence variation is the C-terminal α-helical lid subdomain of substrate-binding domain (SBDα) together with the intrinsically disordered region. Dynamic interactions between the SBDα and β-sandwich substrate-binding subdomain (SBDβ) contribute to the chaperone functions of Hsp70s by tuning kinetics of substrate binding. To investigate how the C-terminal region of Hsp70 has evolved from prokaryotic to eukaryotic organisms, we tested whether this region can be exchanged among different Hsp70 members to support basic chaperone functions. We found that this region from eukaryotic Hsp70 members cannot substitute for the same region in Escherichia coli DnaK to facilitate normal chaperone activity of DnaK. In contrast, this region from E. coli DnaK and Saccharomyces cerevisiae Hsp70 (Ssa1 and Ssa4) can partially support some roles of human stress inducible Hsp70 (hHsp70) and human cognate Hsp70 (hHsc70). Our results indicate that the C-terminal region from eukaryotic Hsp70 members cannot properly support SBDα-SBDβ interactions in DnaK, but this region from DnaK/Ssa1/Ssa4 can still form some SBDα-SBDβ interactions in hHsp70 or hHsc70, which suggests that the mode for SBDα-SBDβ interactions is different in prokaryotic and eukaryotic Hsp70 members. This study provides new insight in the divergency among different Hsp70 homologs and the evolution of Hsp70s.

Contribution of UDP-glycosyltransferases to chlorpyrifos resistance in Nilaparvata lugens

As a multigene superfamily of Phase II detoxification enzymes, uridine diphosphate (UDP)-glycosyltransferases (UGTs) play important roles in the metabolism of xenobiotics including insecticides. In this study, 5-nitrouracil, an inhibitor of UGT enzyme activity, effectively increased the toxicity of chlorpyrifos to the chlorpyrifos-resistant strain of Nilaparvata lugens, one of the most resistant rice pests. The enzyme content of UGT in the resistant strain was significantly higher than that in the susceptible strain. Among 20 identified UGT genes, UGT386H2, UGT386J2, UGT386N2 and UGT386P1 were found significantly overexpressed in the resistant strain and can be effectively induced by chlorpyrifos. These four UGT genes were most highly expressed in the midgut and/or fat body, two main insect detoxification tissues. Amino acid sequence alignments revealed that these four UGTs contained a variable N-terminal substrate-binding domain and a conserved C-terminal sugar donor-binding domain. Furthermore, homology modeling and molecular docking analyses showed that these UGTs could stably bind to chlorpyrifos and chlorpyrifos oxon, with the binding free energies from −19.4 to −110.62 kcal mol−1. Knockdown of UGT386H2 or UGT386P1 by RNA interference dramatically increased the susceptibility of the resistant strain to chlorpyrifos. These findings suggest that overexpression of these two UGT genes contributes to chlorpyrifos resistance in N. lugens.

Emerging enzyme surface display systems for waste resource recovery.

The current century marks an inflection point for human progress, as the developed world increasingly comes to recognize that the ecological and socioeconomic impacts of resource extraction must be balanced with more sustainable modes of growth that are less reliant on non-renewable sources of energy and materials. This has opened a window of opportunity for cross-sector development of biotechnologies that harness the metabolic problem-solving power of microbial communities. In this context, recovery has emerged as an organizing principal to create value from industrial and municipal waste streams, and the search is on for new enzymes and platforms that can be used for waste resource recovery at scale. Enzyme surface display on cells or functionalized materials has emerged as a promising platform for waste valorization. Typically, surface display involves the use of substrate binding or catalytic domains of interest translationally fused with extracellular membrane proteins in a microbial chassis. Novel display systems with improved performance features include S-layer display with increased protein density, spore display with increased resistance to harsh conditions, and intracellular inclusions including DNA-free cells or nanoparticles with improved social licence for in situ applications. Combining these display systems with advances in bioprinting, electrospinning and high-throughput functional screening have potential to transform outmoded extractive paradigms into 'trans-metabolic" processes for remediation and waste resource recovery within an emerging circular bioeconomy.

Exploration of the cysteine reactivity of human inducible Hsp70 and cognate Hsc70

Hsp70s are multifunctional proteins and serve as the central hub of the protein quality control network. Hsp70s are also related to a number of diseases and have been established as drug targets. Human HspA1A (hHsp70) and HspA8 (hHsc70) are the major cytosolic Hsp70s, and they have both overlapping and distinct functions. hHsp70 contains five cysteine residues, and hHsc70 contains four cysteine residues. Previous studies have shown these cysteine residues can undergo different cysteine modifications such as oxidation or reaction with electrophiles to regulate their function, and hHsp70 and hHsc70 have different cysteine reactivity. To address the mechanism of the differences in cysteine reactivity between hHsp70 and hHsc70, we studied the factors that determine this reactivity by Ellman assay for the quantification of accessible free thiols and NMR analysis for the assessment of structural dynamics. We found the lower cysteine reactivity of hHsc70 is probably due to its lower structural dynamics and the stronger inhibition effect of interaction between the α-helical lid subdomain of the substrate-binding domain (SBDα) and the β-sheet substrate-binding subdomain (SBDβ) on cysteine reactivity of hHsc70. We determined that Gly557 in hHsp70 contributes significantly to the higher structural dynamics and cysteine reactivity of hHsp70 SBDα. Exploring the cysteine reactivity of hHsp70 and hHsc70 facilitates an understanding of the effects of redox reactions and electrophiles on their chaperone activity and regulation mechanisms, and how these differences allow them to undertake distinct cellular roles.

Abstract A080: METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8

Abstract Background: Our work focuses on the role of METTL21A, a member of the little studied seven β-strand family of candidate lysine methyltransferases (KMTs), and its role in the regulation of pancreatic ductal adenocarcinoma (PDAC) development. Using a meta-analysis of publicly available gene expression datasets, we found that METTL21A is significantly downregulated in PDAC versus normal tissue and the reduced expression predicts poor patient survival. Thus, there are intriguing correlations connecting METTL21A to PDAC pathogenesis; however, to date, there are no data on the role of METTL21A in cancer and an enzymatic function for METTL21A is poorly recognized. Methods: To test the hypothesis that METTL21A plays a role in PDAC initiation and progression, we generated conditional Mettl21a knockout mice and crossed to PDAC models: p48Cre/+ KrasG12D/+ (Kras vs. Kras;Mettl21a) and p48Cre/+ KrasG12D/+ p53fl/fl (Kras;p53 vs. Kras;p53;Mettl21a). Furthermore, we studied the impact of METTL21A depletion on human PDAC cell lines and PDX growth in vivo and in vitro. To identify the substrate of METTL21A enzymatic activity we performed methylation assays followed by mass spectrometry analysis. Results: Histopathological analysis of pancreatic tissues of Kras and Kras;Mettl21a mice at 6 months of age revealed that loss of METTL21A significantly accelerates pancreatic cancer initiation. Additional studies using Kras;p53 and Kras;p53;Mettl21a mice demonstrated that METTL21A depletion accelerates PDAC progression and results in a significantly shorter overall survival. In congruence with those observations, knockdown of METTL21A in human PDAC cell lines leads to the robust increase in cell proliferation, enhances colony formation ability in vitro and xenograft growth in vivo. In contrast, cells rescued with ectopic expression of wildtype METTL21A but not with enzyme-dead mutant METTL21A showed significant growth impairment in vitro and in vivo. Next, our in vivo methylation assays identified HSPA1 and HSPA8 (members of the HSP70 protein family) as substrates of METTL21A in PDAC cells. Our subsequent analysis showed that METTL21a specifically tri-methylates HSPA1 and HSPA8 at lysine 561 (K561me3). Next, to directly evaluate the role of METTL21A mediated methylation of HSPA1/A8 at K561 in regulating cancer phenotype, we knocked out HSPA1/A8 and complemented with expression of wildtype or K561A mutant HSPA1/A8 in PDAC cell lines. We found that only methylation-resistant HSPA1/A8 increased cell proliferation and tumor growth in vivo. Together, these results argue that in PDAC, the principal physiologic activity of METTL21A is generation of HSPA1/A8 K561me3, which suppresses cancer growth. Conclusion: Our research revealed that METTL21A is a novel tumor suppressor that inhibits the initiation and progression of PDAC through methylation of the heat-shock proteins HSPA1/8 substrate-binding domain. Citation Format: Xiaojie Yang, Mohamad Zoabi, Simone Hausmann, Natasha M. Flores, Xiaoyin Lu, Jibo Wu, Ana Morales-Benitez, Or Gozani, Pawel K. Mazur. METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A080.

ERK2 Substrate Binding Domains Play Distinct Roles in Megakaryocytic-Erythroid Lineage Progression and Mediates Clonal Fitness in Myeloproliferative Neoplasms

ERK2 Substrate Binding Domains Play Distinct Roles in Megakaryocytic-Erythroid Lineage Progression and Mediates Clonal Fitness in Myeloproliferative Neoplasms

Mutation of FMDV Lpro H138 residue drives viral attenuation in cell culture and in vivo in swine

The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) is a papain like protease that cleaves the viral polyprotein and several host factors affecting host cell translation and induction of innate immunity. Introduction of Lpro mutations ablating catalytic activity is not tolerated by the virus, however, complete coding sequence deletion or introduction of targeted amino acid substitutions can render viable progeny. In proof-of-concept studies, we have previously identified and characterized FMDV Lpro mutants that are attenuated in cell culture and in animals, while retaining their capacity for inducing a strong adaptive immunity. By using molecular modeling, we have now identified a His residue (H138), that resides outside the substrate binding and catalytic domain, and is highly conserved across serotypes. Mutation of H138 renders possible FMDV variants of reduced virulence in vitro and in vivo. Kinetics studies showed that FMDV A12-LH138L mutant replicates similarly to FMDV A12-wild type (WT) virus in cells that do not offer immune selective pressure, but attenuation is observed upon infection of primary or low passage porcine epithelial cells. Western blot analysis on protein extracts from these cells, revealed that while processing of translation initiation factor eIF-4G was slightly delayed, no degradation of innate sensors or effector molecules such as NF-κB or G3BP2 was observed, and higher levels of interferon (IFN) and IFN-stimulated genes (ISGs) were induced after infection with A12-LH138L as compared to WT FMDV. Consistent with the results in porcine cells, inoculation of swine with this mutant resulted in a mild, or in some cases, no clinical disease but induction of a strong serological adaptive immune response. These results further support previous evidence that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.

Capturing intrinsic nanomechanics of allostery

The Hsp70 chaperone exploits allosteric communication between its substrate binding domain and its nucleotide binding domain to regulate the loading and release of misfolded polypeptides in an ATP-hydrolysis-dependent manner. In this issue of Biophysical Journal, Singh, Rief, and Žoldák report an exquisitely detailed study of the nanomechanical aspects of the allosteric mechanism in DnaK, an Escherichia coli heat shock protein 70 chaperone.