More Like this

1. The C-Terminus of Perilipin 3 Shows Distinct Lipid Binding at Phospholipid-Oil-Aqueous Interfaces

   https://doi.org/10.3390/membranes11040265  

   Titus, Amber R. ; Ridgway, Ellyse N. ; Douglas, Rebecca ; Brenes, Elena Sánchez ; Mann, Elizabeth K. ; Kooijman, Edgar E. ( April 2021 , Membranes)

   null (Ed.)

   Lipid droplets (LDs) are ubiquitously expressed organelles; the only intracellular organelles that contain a lipid monolayer rather than a bilayer. Proteins localize and bind to this monolayer as they do to intracellular lipid bilayers. The mechanism by which cytosolic LD binding proteins recognize, and bind, to this lipid interface remains poorly understood. Amphipathic α-helix bundles form a common motif that is shared between cytosolic LD binding proteins (e.g., perilipins 2, 3, and 5) and apolipoproteins, such as apoE and apoLp-III, found on lipoprotein particles. Here, we use pendant drop tensiometry to expand our previous work on the C-terminal α-helix bundle of perilipin 3 and the full-length protein. We measure the recruitment and insertion of perilipin 3 at mixed lipid monolayers at an aqueous-phospholipid-oil interface. We find that, compared to its C-terminus alone, the full-length perilipin 3 has a higher affinity for both a neat oil/aqueous interface and a phosphatidylcholine (PC) coated oil/aqueous interface. Both the full-length protein and the C-terminus show significantly more insertion into a fully unsaturated PC monolayer, contrary to our previous results at the air-aqueous interface. Additionally, the C-terminus shows a preference for lipid monolayers containing phosphatidylethanolamine (PE), whereas the full-length protein does not. These results strongly support a model whereby both the N-terminal 11-mer repeat region and C-terminal amphipathic α-helix bundle domains of perilipin 3 have distinct lipid binding, and potentially biological roles. 

   more » « less
2. Mechanical properties of BiP protein determined by nano‐rheology

   https://doi.org/10.1002/pro.3432  

   Casanova‐Morales, Nathalie ; Quiroga‐Roger, Diego ; Alfaro‐Valdés, Hilda M. ; Alavi, Zahra ; Lagos‐Espinoza, Miguel I. A. ; Zocchi, Giovanni ; Wilson, Christian A. M. ( September 2018 , Protein Science)

   Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N‐terminal Nucleotide‐Binding Domain (NBD), and the C‐terminal Substrate‐Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano‐rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides‐ ATP, ADP, and AMP‐PNP‐, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (K_D) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.

    

   more » « less
3. Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

   https://doi.org/10.1038/s42003-021-02924-2  

   Williams, Leah M. ; Sridhar, Sainetra ; Samaroo, Jason ; Peart, Jada ; Adindu, Ebubechi K. ; Addanki, Anvitha ; DiRusso, Christopher J. ; BB522 Molecular Biology Laboratory ; Alburi, Dana H. M. ; Anisimov, Ludmila ; et al ( December 2021 , Communications Biology)

   We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoanCapsaspora owczarzaki(Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allowsCo-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing ofCo-NF-κB is not induced by IκB kinases in human cells. OverexpressedCo-NF-κB localizes to the cytoplasm inCocells.Co-NF-κB mRNA and DNA-binding levels differ across threeCapsasporalife stages. RNA-sequencing and GO analyses identify possible gene targets ofCo-NF-κB. Three NF-κB-like proteins from the choanoflagellateAcanthoeca spectabilis(As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All threeAs-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.

    

   more » « less
4. Intra- and inter-molecular regulation by intrinsically-disordered regions governs PUF protein RNA binding

   https://doi.org/10.1038/s41467-023-43098-1  

   Qiu, Chen ; Zhang, Zihan ; Wine, Robert N ; Campbell, Zachary T ; Zhang, Jun ; Hall, Traci_M Tanaka ( December 2023 , Nature Communications)

   PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities.Caenorhabditis elegansPUF proteinfem-3binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5′ end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal.

    

   more » « less
5. A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions

   https://doi.org/10.1111/mmi.13947  

   Blank, Matthew L. ; Parker, Michelle L. ; Ramaswamy, Raghavendran ; Powell, Cameron J. ; English, Elizabeth D. ; Adomako‐Ankomah, Yaw ; Pernas, Lena F. ; Workman, Sean D. ; Boothroyd, John C. ; Boulanger, Martin J. ; et al ( April 2018 , Molecular Microbiology)

   TheToxoplasma gondiilocusmitochondrial association factor 1(MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose inT. gondiithrough neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA‐competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro‐domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C‐terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C‐terminal helix are required for MAF1‐mediated HMA. Importantly these same three residues are also required for thein vivogrowth advantage conferred by MAF1b, providing a definitive link betweenin vivoproliferation and manipulation of host mitochondria. Co‐immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA‐competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversifiedT. gondiilocus.

    

   more » « less