Low complexity protein sequences are often intrinsically unstructured and many have the potential to polymerize into amyloid aggregates including filaments and hydrogels. RNA-binding proteins are unusually enriched in such sequences raising the question as to what function these domains serve in RNA metabolism. One such yeast protein, Nab3, is an 802 amino acid termination factor that contains an RNA recognition motif and a glutamine/proline rich domain adjacent to a region with structural similarity to a human hnRNP. A portion of the C-terminal glutamine/proline-rich domain assembles into filaments that organize into a hydrogel. Here we analyze the determinants of filament formation of the isolated low complexity domain as well as examine the polymerization properties of full-length Nab3. We found that the C-terminal region with structural homology to hnRNP-C is not required for assembly, nor is an adjacent stretch of 16 glutamines. However, reducing the overall glutamine composition of this 134-amino acid segment from 32% to 14% destroys its polymerization ability. Importantly, full-length wildtype Nab3 also formed filaments with a characteristic cross-β structure which was dependent upon the glutamine/proline-rich region. When full length Nab3 with reduced glutamine content in its low complexity domain was exchanged for wildtype Nab3, cells were not viable. This suggests that polymerization of Nab3 is normally required for its function. In an extension of this idea, we show that the low complexity domain of another yeast termination factor, Pcf11, polymerizes into amyloid fibers and a hydrogel. These findings suggest that, like many other RNA binding proteins, termination factors share a common biophysical trait that may be important for their function.
Evidence suggests that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, may reduce the risk of Alzheimer's disease (AD). Statin action in patients with AD, as in those with heart disease, is likely to be at least partly independent of the effects of statins on cholesterol. Statins can alter cellular signaling and protein trafficking through inhibition of isoprenylation of Rho, Cdc42, and Rab family GTPases. The effects of statins on protein isoprenylation in vivo, particularly in the central nervous system, are poorly studied. We utilized two-dimensional gel electrophoresis approaches to directly monitor the levels of isoprenylated and non-isoprenylated forms of Rho and Rab family GTPases. We report that simvastatin significantly inhibits RhoA and Rab4, and Rab6 isoprenylation at doses as low as 50 nM in vitro. We also provide the first in vivo evidence that statins inhibit the isoprenylation of RhoA in the brains of rats and RhoA, Cdc42, and H-Ras in the brains of mice treated with clinically relevant doses of simvastatin.
KGaA, Weinheim Protein–ligand interactions serve as fundamental regulators of numerous biological processes. Among protein–ligand pairs, glycan binding proteins (GBPs) and the glycans they recognize represent unique and highly complex interactions implicated in a broad range of regulatory activities. With few exceptions, cell surface receptors and secreted proteins are heavily glycosylated. As these glycans often represent highly regulatable post-translational modifications, alterations in glycosylation can fundamentally impact GBP recognition. Among GBPs, galectins in particular appear to engage a diverse set of glycan determinants to impact a broad range of biological processes. In this review, we will explore factors that impact galectin activity, including the effect of glycan modification on galectin–glycan interactions.
Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys, and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBPs) expressed by dendritic cells (DC) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and Siglecs expressed by DC for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-SIGN showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglecs-5 and -9 showed weak binding to a few glycans. By contrast, most hGBPs bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ~1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBPs expressed by DC binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant.
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John Horton;
Xu Liu;
Molly Gale;
Lizhen Wu;
John R. Shanks;
Xing Zhang;
Philip J. Webber;
Joshua S.K. Bell;
Stephen C Kales;
Bryan T Mott;
Ganesha Rai;
Daniel J Jansen;
Mark J Henderson;
Daniel J Urban;
Matthew D Hall;
Anton Simeonov;
David J Maloney;
Margaret A. Johns;
Haian Fu;
Ajit Jadhav;
Paula Vertino;
Qin Yan;
Xiaodong Cheng
The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases removes methyl groups from methylated lysine 4 of histone H3. Accumulating evidence supports a role for KDM5 family members as oncogenic drivers. We compare the in vitro inhibitory properties and binding affinity of ten diverse compounds with all four family members, and present the crystal structures of the KDM5A-linked Jumonji domain in complex with eight of these inhibitors in the presence of Mn(II). All eight inhibitors structurally examined occupy the binding site of α-ketoglutarate, but differ in their specific binding interactions, including the number of ligands involved in metal coordination. We also observed inhibitor-induced conformational changes in KDM5A, particularly those residues involved in the binding of α-ketoglutarate, the anticipated peptide substrate, and intramolecular interactions. We discuss how particular chemical moieties contribute to inhibitor potency and suggest strategies that might be utilized in the successful design of selective and potent epigenetic inhibitors.
Over the last quarter-century, there has been tremendous progress in genetics research that has defined molecular causes for cardiomyopathies. More than a thousand mutations have been identified in many genes with varying ontologies, therein indicating the diverse molecules and pathways that cause hypertrophic, dilated, restrictive, and arrhythmogenic cardiomyopathies. Translation of this research to the clinic via genetic testing can precisely group affected patients according to molecular etiology, and identify individuals without evidence of disease who are at high risk for developing cardiomyopathy. These advances provide insights into the earliest manifestations of cardiomyopathy and help to define the molecular pathophysiological basis for cardiac remodeling. Although these efforts remain incomplete, new genomic technologies and analytic strategies provide unparalleled opportunities to fully explore the genetic architecture of cardiomyopathies. Such data hold the promise that mutation-specific pathophysiology will uncover novel therapeutic targets, and herald the beginning of precision therapy for cardiomyopathy patients.
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Annette Ehrhardt;
Wook Chung;
Louise C. Pyle;
Wei Wang;
Krzysztof Nowotarski;
Cory M. Mulvihill;
Mohabir Rannjeesingh;
Jeong Hong;
Sadanandan E. Velu;
Hal A. Lewis;
Shane Atwell;
Steve Aller;
Christine E. Bear;
Gergely L. Lukacs;
Kevin L. Kirk;
Eric Sorscher
In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.
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Jairo Andres Fonseca;
Monica Cabrera-Mora;
Balwan Singh;
Joseli Oliveira-Ferreira;
Josué da Costa Lima-Junior;
J. Mauricio Calvo-Calle;
Jose Manuel Manuel Lozano;
C. Moreno
The most widespread Plasmodium species, Plasmodium vivax, poses a significant public health threat. An effective vaccine is needed to reduce global malaria burden. Of the erythrocytic stage vaccine candidates, the 19 kDa fragment of the P. vivax Merozoite Surface Protein 1 (PvMSP119) is one of the most promising. Our group has previously defined several promiscuous T helper epitopes within the PvMSP1 protein, with features that allow them to bind multiple MHC class II alleles. We describe here a P. vivax recombinant modular chimera based on MSP1 (PvRMC-MSP1) that includes defined T cell epitopes genetically fused to PvMSP119. This vaccine candidate preserved structural elements of the native PvMSP119 and elicited cytophilic antibody responses, and CD4+ and CD8+ T cells capable of recognizing PvMSP119. Although CD8+ T cells that recognize blood stage antigens have been reported to control blood infection, CD8+ T cell responses induced by P. falciparum or P. vivax vaccine candidates based on MSP119 have not been reported. To our knowledge, this is the first time a protein based subunit vaccine has been able to induce CD8+ T cell against PvMSP119. The PvRMC-MSP1 protein was also recognized by naturally acquired antibodies from individuals living in malaria endemic areas with an antibody profile associated with protection from infection. These features make PvRMC-MSP1 a promising vaccine candidate.
A discovery study was carried out where serum samples from 22 systemic lupus erythematosus (SLE) patients and matched healthy controls were hybridized to antibody-coated glass slide arrays that interrogated the level of 274 human proteins. On the basis of these screens, 48 proteins were selected for ELISA-based validation in an independent cohort of 28 SLE patients. Whereas AXL, ferritin, and sTNFRII were significantly elevated in patients with active lupus nephritis (LN) relative to SLE patients who were quiescent, other molecules such as OPN, sTNFRI, sTNFRII, IGFBP2, SIGLEC5, FAS, and MMP10 exhibited the capacity to distinguish SLE from healthy controls with ROC AUC exceeding 90%, all with p < 0.001 significance. These serum markers were next tested in a cohort of 45 LN patients, where serum was obtained at the time of renal biopsy. In these patients, sTNFRII exhibited the strongest correlation with eGFR (r = −0.50, p = 0.0014) and serum creatinine (r = 0.57, p = 0.0001), although AXL, FAS, and IGFBP2 also correlated with these clinical measures of renal function. When concurrent renal biopsies from these patients were examined, serum FAS, IGFBP2, and TNFRII showed significant positive correlations with renal pathology activity index, while sTNFRII displayed the highest correlation with concurrently scored renal pathology chronicity index (r = 0.57, p = 0.001). Finally, in a longitudinal cohort of seven SLE patients examined at ∼3 month intervals, AXL, ICAM-1, IGFBP2, SIGLEC5, sTNFRII, and VCAM-1 demonstrated the ability to track with concurrent disease flare, with significant subject to subject variation. In summary, serum proteins have the capacity to identify patients with active nephritis, flares, and renal pathology activity or chronicity changes, although larger longitudinal cohort studies are warranted.