Journal of Biological Chemistry

N-terminal protein methylation (N-methylation) is a post-translational modification (PTM) that influences a variety of biological processes by regulating protein stability, protein-DNA interactions, and protein-protein interactions. Although significant progress has been made in understanding the biological roles of this PTM, we still do not completely understand how the methyltransferases that place it are regulated. A common mode of methyltransferase regulation is through complex formation with close family members, and we have previously shown that the N-trimethylase METTL11A (NRMT1/NTMT1) is activated through binding of its close homolog METTL11B (NRMT2/NTMT2). It has also recently been reported that METTL11A co-fractionates with a third METTL family member METTL13, which methylates both the N-terminus and lysine 55 (K55) of eukaryotic elongation factor 1 alpha (eEF1A). Here we confirm a regulatory interaction between METTL11A and METTL13 and show that, while METTL11B is an activator of METTL11A, METTL13 inhibits METTL11A activity. This is the first example of a methyltransferase being opposingly regulated by different family members. Similarly, we find that METTL11A promotes the K55 methylation activity of METTL13 but inhibits its N-methylation activity. We also find that catalytic activity is not needed for these regulatory effects, demonstrating new, non-catalytic functions for METTL11A and METTL13. Finally, we show METTL11A, METTL11B, and METTL13 can complex together, and when all three are present, the regulatory effects of METTL13 take precedence over those of METTL11B. These findings provide a better understanding of the regulation of N-methylation, and suggest a model where these methyltransferases can serve in both catalytic and non-catalytic roles.

Cellular plasminogen (Pg) receptors (PgR) are utilized to recruit Pg, stimulate its activation to the serine protease, plasmin (Pm), and sterically protect the generated Pm from inactivation by natural host inhibitors. The net result is that cells contain a stable proteolytic surface used for biological mechanisms involved in cell migration. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this regard. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 [A] as a stable symmetrical homooctamer displaying point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ~49 kDa. Subunit-subunit interactions showed four major and four minor interfaces in the octamer. Binding sites for hPg were previously proposed to include the COOH-terminal K434,435 residues of Sen, but in native Sen these residues are buried within the minor interfaces of the octamer and do not function as a Pg binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K434,434. We propose that the octameric structure of Sen is important to its ability to interact with hPg, but disruption of its overall octameric conformation without dissociation of the octamer exposes neoepitopes for hPg binding.

Tau aggregation is a key pathological feature of neurodegenerative diseases termed tauopathies. Identifying the various cellular factors that function to prevent tau aggregation in cells can generate key insights into how to mitigate diseases associated with protein misfolding. During an investigation into developing purification methods for the protein tau, we observed that isolates of E. coli lysate prevented human tau aggregation in vitro. Fractionation of the lysate was used to further isolate a small molecular weight (MW) inhibitory fraction containing multiple components, as determined by mass spectrometry and NMR. A putative inhibitory component, methylphosphonic acid (MePn), decreased tau amyloid formation when supplemented to in vitro aggregation assays. MePn also blocked the aggregation of expressed tau in live E. coli when supplemented to the culture media. Our findings can be directly applied to optimizing purification of recombinant tau protein and more broadly, highlight the potential of cellular metabolites to directly modulate tau amyloid formation.

Heme-regulated inhibitor (HRI) is a key modulator of hemoglobin synthesis, exerting such effects by sensing intracellular heme levels. Under heme-deficient conditions, HRI dissociates from heme, becomes activated, and phosphorylates the translation initiation factor eIF2. However, the precise regulatory mechanisms governing HRI activation remain incompletely understood. In this study, we delineate part of the regulatory mechanism involving autophosphorylation-dependent activation and heme-mediated deactivation of HRI. HRI formed a dimer in solution through its N-terminal domain, irrespective of its phosphorylation state. However, a mutant with deletion of the N-terminal domain retained autophosphorylation activity, indicating that N-terminal domain-mediated dimerization is not essential for activation. Phosphorylated HRI formed a stable complex with eIF2, whereas the dephosphorylated form failed to bind, indicating that autophosphorylation is required for eIF2 recognition. Biochemical analyses, together with modeling based on the predicted structure, revealed that the phosphate groups of Thr488 and Thr493 interact with adjacent basic residues. These interactions enhance eIF2 recognition and phosphorylation. Additionally, heme-induced deactivation selectively targeted the dephosphorylated kinase domain and suppressed autophosphorylation. These findings provide multiple mechanistic insights into regulation of the activity of HRI, highlighting distinct roles for autophosphorylation, structural elements, and heme responsiveness in controlling its function.

Human transmembrane proteins Serinc3 and Serinc5 are antiviral restriction factors that inhibit HIV-1 infectivity. In the absence of viral antagonism, Serinc3 and Serinc5 incorporate into the envelopes of nascent virions and inhibit the fusion of virions to the target cells. The HIV-1 virus counteracts the restriction of Serinc3 by downregulating it from the cell surface and thus excluding it from budding virions. This is orchestrated by the viral accessory protein Nef and involves hijacking of the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. The mechanistic details of Nef-mediated Serinc3 downregulation, however, have been enigmatic. In this work, we investigated and revealed the molecular determinants of Serinc3 modulation by Nef. Our results show that Nef recruits Serinc3 by binding to its N-terminal cytosolic tail. Furthermore, Nef residues important for Serinc3-binding in vitro, and for the exclusion of Serinc3 from virions, overlap with those required for Nef-mediated CD4 downregulation, suggesting great mechanistic similarities between the two functions of Nef. In addition to shedding light to the mechanism of Serinc3 antagonism, our work also highlights the conserved substrate-binding pocket of Nef as a molecular hotspot for inhibitor development and antiretroviral drug discovery.

A common feature among nearly all Gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate. Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg2+ as a cofactor. Mn2+, but not other divalent cations, could substitute for Mg2+. Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD-C6-ceramide (Km,app=19.2 {+/-} 5.5 M; Vmax,app=2586.29 {+/-} 231.99 pmol/min/mg enzyme) and ATP (Km,app=0.29 {+/-} 0.07 mM; Vmax,app=10067.57 {+/-} 996.85 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. ADP-ribosylation results from the transfer of the ADP-ribose moiety of NAD+ to various acceptors, e.g., proteins, DNA, RNA, contributing on a cells biological processes. ADP-ribosylation is the mechanism of action of bacterial toxins, e.g., Pseudomonas toxins, diphtheria toxin that disrupt protein biosynthetic and signaling pathways. On the other hand, some viral macrodomains cleavage ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved -NAD+, but not {beta}-NAD+, consistent with stereospecificity at the C-1" bond. In contrast to ARH1 and ARH3, Mac1 did not require Mg2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1"-phosphat, but not Mac2 and Mac3. However, Mac1 did not cleave -ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 activities may shed light on potential SARS-CoV-2 therapeutic targets. IMPORTANCESARS-CoV-2, the virus responsible for COVID-19, encodes 3 macrodomain-containing proteins, e.g., Mac1, Mac2, Mac3, within non-structural proteins 3 (Nsp3). Mac1 was shown previously to hydrolyze ADP-ribose-phosphate. Inactivation of Mac1 reduced viral proliferation. Here we report that Mac1, but not Mac2 and Mac3, has multiple activities, i.e., Mac1 hydrolyzed. -NAD+ and O-acetyl-ADP-ribose. However, Mac1 did not hydrolyze {beta}-NAD+, ADP-ribose-serine on a histone 3 peptide (aa1-21), and ADP-ribose-arginine, exhibiting substrate selectivity. These data suggest that Mac1 may have multi-function as a -NAD+ consumer for viral replication and a disruptor of host-mediated ADP-ribosylation pathways. Understanding Mac1s mechanisms of action is important to provide possible therapeutic targets for COVID-19.

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (virus inhibitory protein endoplasmic reticulum-associated, interferon-induced) is an ER membrane-associated enzyme that when expressed in response to viral infections exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. Here we have investigated the effect of viperin expression on cholesterol biosynthesis. We found that viperin expression reduces cholesterol levels by 20 - 30 % in HEK293T cells. A proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits. The two most highly enriched proteins were lanosterol synthase and squalene monooxygenase, enzymes that catalyze key steps establishing the sterol carbon skeleton. Co-immunoprecipitation experiments established that viperin, lanosterol synthase and squalene monooxygenase form a complex at the ER membrane. Co-expression of viperin was found to significantly inhibit the specific activity of lanosterol synthase in HEK293T cell lysates. Co-expression of viperin had no effect on the specific activity of squalene monooxygenase, but reduced its expression levels in the cells by approximately 30 %. Despite these inhibitory effects, co-expression of either LS or SM failed to reverse the viperin-induced depletion of cellular cholesterol levels in HEK293T cells. Our results establish a clear link between the down-regulation of cholesterol biosynthesis and viperin, although at this point the effect cannot be unambiguously attributed interactions between viperin and a specific biosynthetic enzyme.

-catenin binds directly to {beta}-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates -catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of T(testes)-catenin, an -catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that T-catenin binds the {beta}-catenin/N-cadherin complex with a similar low nanomolar affinity to that of E-catenin. Limited proteolysis revealed that the T-catenin M-region adopts a more open conformation than E-catenin. The T-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated T-catenin M-region is not autoinhibited and thereby distinct from E-catenin. However, the T-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, T-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results indicate that the T-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of T-catenin allow it to function as a scaffold for building specific adhesion complexes.

Lipopolysaccharide (LPS), a ubiquitous bacterial component of food, is neutralized by a variety of mechanisms that help to establish a threshold, which when exceeded results in an inflammatory TLR4 mediated response. Notably both CEACAM1 and CD36 affect downstream signaling of TLR4 to LPS. Furthermore, CEACAM1 associates with CD36 in hepatocytes, regulating lipid storage and bile acid (BA) secretion that includes reverse transport of LPS to the intestine. Direct binding of LPS-Ra micelles to soluble CEACAM1 or soluble CD36 was analyzed by surface plasmon resonance (SPR), size exclusion chromatography (SEC) and transmission electron microscopy (TEM). Direct binding of CEACAM1 to CD36 was analyzed by SPR and proximity ligation assays. Molecular models were generated by Alpha Fold and Molecular Dynamics. LPS Binding: SPR binding constants of KD= 1.04 x 10-10 M and KD= 3.38 x 10-10 M were obtained for LPS-Ra micelle binding to sCEACAM1 and sCD36, respectively. On SEC, the molecular sizes of LPS-Ra micelles bound to sCEACAM1 and sCD36 were approximately 500 and 800 kDa, respectively. In addition, LPS binding to both was reduced by sodium cholate and sodium deoxycholate. Alpha Fold predicted a binding site of LPS-Ra to CD36, while Molecular Dynamic studies of an N-domain mutant of CEACAM1, that breaks a conserved salt bridge, revealed the presence of an open form that is predicted to bind LPS. sCEACAM1 to sCD36 Binding: A KD of 5.28 x 10-8 M was obtained for sCEACAM1 binding to immobilized sCD36 by SPR. Antibody-based-proximity ligation demonstrated the association of the ectodomains of CEACAM1 and CD36 on hepatic cells and when co-expressed in HEK cells. In addition, biotin-based proximity ligation demonstrated association of the cytoplasmic domains of CEACAM1 and a CD36-BioID2 fusion protein when co-expressed in HEK cells. Alpha Fold predicted both head-to-head (trans) and side-to-side (cis) binding of the N-domain of CEACAM1 to CD36, from which a membrane model of their cis-interaction could account for the proximity ligation results. Both CEACAM1 and CD36 share a common LPS micelle binding function, as well as binding to each other, and together, may regulate uptake and excretion of micellar LPS.

Hepatic tryptophan 2,3-dioxygenase (TDO) is a cytoplasmic homotetrameric hemoprotein and the rate-limiting enzyme in the irreversible degradation of the essential amino acid L-tryptophan (L-Trp) to N-formylkynurenine, thus controlling the flux of L-Trp into its serotonergic and kynureninic/NAD pathways. TDO has long been recognized to be substrate-inducible via protein stabilization, but the molecular mechanism of this stabilization has remained elusive. Recent elucidation of human TDO (hTDO) crystal structure has identified a high-affinity (Kd {approx} 0.5 M) Trp-binding exosite in each of its 4 monomeric subunits. Mutation of the Glu105, Trp208 and Arg211 comprising this exosite not only abolished the high-affinity L-Trp binding, but also accelerated the ubiquitin-dependent proteasomal degradation of hTDO. We have further characterized this hTDO degradation by documenting that its ubiquitination by gp78/AMFR and CHIP E2/E3 ligase complexes occurs on external Lys-residues within or vicinal to acidic Asp/Glu and phosphorylated pSer/pThr (DEpSpT)-clusters. Furthermore, we have identified the unstructured hTDO N- and C-termini as imparting relatively high proteolytic instability, as their deletion ({Delta}NC) markedly prolonged hTDO t1/2. Additionally, although previous studies reported that upon hepatic heme-depletion, the heme-free apoTDO turns over with a t1/2 {approx} 2.2 h relative to the t1/2 of 7.7 h of holoTDO, mutating the axial heme-ligating His328 to Ala has the opposite effect of prolonging hTDO t1/2. Most importantly, introducing the exosite mutation into the {Delta}NC-deleted or H328A-mutant completely abolished their prolonged half-lives irrespective of L-Trp presence or absence, thereby revealing that the exosite is the molecular lynchpin that defines L-Trp-mediated TDO induction via protein stabilization.

Protein kinase A (PKA) is a crucial signaling enzyme in neurons, with its dysregulation being implicated in neurodegenerative diseases. Assembly of the PKA holoenzyme, comprising a dimer of heterodimers of regulatory (R) and catalytic (C) subunits, ensures allosteric regulation and functional specificity. Recently, we defined the RI{beta}-L50R variant as a causative mutation that triggers protein aggregation in a rare neurodegenerative disease. However, the mechanism underlying uncontrolled PKA allosteric regulation and its connection to the functional outcomes leading to clinical symptoms remain elusive. In this study, we established an in vitro model using patient-derived cells for a personalized approach and employed direct measurements of purified proteins to investigate disease mechanisms in a controlled environment. Structural analysis and circular dichroism spectroscopy revealed that cellular proteins aggregation resulted from misfolded RI{beta}-subunits, preventing holoenzyme assembly and anchoring through A Kinase Anchoring Proteins (AKAPs). While maintaining high affinity to the C subunit, the resulting RI{beta}-L50R:C heterodimer exhibits reduced cooperativity, requiring lower cAMP concentrations for dissociation. Consequently, there was an increased translocation of C-subunit into the nucleus, impacting gene expression. We successfully controlled C subunit translocation by introducing a mutation that decreased RI{beta}:C dissociation in response to elevated cAMP levels. This research thus sets the stage for developing therapeutic strategies that modulate PKA assembly and allostery, thus exerting control over the unique molecular signatures identified in the disease-associated transcriptome profile.

Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KD = 5.64 {micro}M) but not the ferrous (Fe2+) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H2S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS-Fe3+. These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase. Significance StatementHydrogen sulfide (H2S) is an important signaling molecule in eukaryotes and bacteria, and along with CO and NO, is an important part of host defense against Mycobacterium tuberculosis (Mtb). However, the mechanism(s) by which Mtb senses and responds to H2S is unknown. Here, we report that the Mtb heme sensor kinase DosS, a known sensor of CO and NO, is also a sensor of H2S. We found that H2S binds DosS in its ferric (Fe3+) state, which is considered as its inactive state, to induce the Dos dormancy regulon during infection. These data highlight the unusual capacity of Mtb to sense multiple gasotransmitters through a single sensing protein.

Metals are widespread environmental toxins that disrupt normal cellular processes through their interactions with proteins and other macromolecules. In this study, we developed the metalsensitive fluorescent protein mseGFP as a ratiometric reporter capable of binding heavy metals. We found that mseGFP bound mercury and lead tightly but had substantially lower sensitivity to other metals. By comparison, the redox sensor roGFP2 functioned as a ratiometric indicator for transition metals, with the highest sensitivity for copper, followed by nickel and cobalt. mseGFP and roGFP2 could also report metal binding through fluorescence quenching, and we used this effect to measure high affinity interactions for both proteins with copper and iron. Crystal structure analysis of mseGFP complexed with phenylarsine oxide revealed an unexpected mode of heavy metal interaction, with mseGFP binding PAO with 2:2 stoichiometry. Glutathione strongly inhibited most metal interactions with the fluorescent protein reporters, but increased the affinity of arsenic and cadmium for mseGFP. When expressed in HEK293T cells, mseGFP reported uptake of mercury and phenylarsine oxide from surrounding media. Glutathione depletion enhanced binding of phenylarsine oxide to mseGFP in cells, validating the importance of glutathione in modulating metal-protein interactions.

The cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. Full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein (+TIP) CLIP-170 has been implicated in this actin-MT coordination by associating with the actin-binding signaling protein IQGAP1, and by promoting actin polymerization through binding with formins. Thus far, CLIP-170s interactions with actin were assumed to be indirect. Here, we demonstrate that CLIP-170 can bind to filamentous actin (F-actin) directly. The affinity is relatively weak, but is strong enough to be significant in the actin-rich cortex, where actin concentrations can be extremely high. Using CLIP-170 fragments and mutants, we show that the direct CLIP-170:actin interaction is independent of the FEED domain, the region that mediates formin-dependent actin polymerization, and that the CLIP-170 F-actin-binding region overlaps with the MT-binding region. Consistent with these observations, in vitro competition assays indicate that CLIP-170:F-actin and CLIP-170:MT interactions are mutually exclusive. Taken together, these observations lead us to speculate that direct CLIP-170:F-actin interactions may function to reduce the stability of MTs in actin-rich regions of the cell, as previously proposed for EB1.

Soluble type-M immunoglobulins (IgMs), among the most potent activators of the classical pathway, are key mediators of complement-dependent cytotoxicity, which render them promising drug candidates for the development of alternative drugs in treating autoimmune or inflammatory diseases. In this study, we investigated the biochemical and in vitro functional properties of recombinant fragments from IgMs corresponding to the Fc-core in their pentameric or hexameric forms. Biophysical experiments confirmed the crucial role of the IgM Joining chain (J) in favoring homogenous pentamers, while its absence led to heterogeneous population with a mixture of oligomeric forms. By combining size-exclusion chromatography with mass photometry, isolation of enriched samples with IgM hexamers or IgM pentamers without the J chain was possible. Biolayer interferometry demonstrated that both IgM-Fc forms bind C1q and ELISA showed that they induce the in vitro C4b deposition when in solid phase. Additionally, our data confirmed the higher efficacy of IgM hexamers compared to pentamers in activating the first component of the classical pathway. Finally, hemolytic assays demonstrate the ability of IgM-Fc constructs to inhibit Ig-induced complement-dependent cytotoxicity, which is likely made possible by the absence of Fab. These findings suggest a possible mechanism of C1 sequestration in plasma by IgM cores and consumption of the initial complement component C4. Our data thus provide important information for the development of IgM-based anti-inflammatory molecules that target specifically complement activation.

-Synuclein (Syn) is a small (140 amino acids) disordered, acidic (pI: 4.7) protein, highly conserved in vertebrates and implicated in the pathogenesis of Parkinsons disease (PD), a neurodegenerative disease characterized by the deposition of Syn amyloid fibrils in dopaminergic neurons. Beyond the central nervous system, significant expression of Syn has also been measured in the blood (~1 M), where platelets are the main cellular hosts of Syn. Although the pathological implication of Syn in PD is widely accepted, the physiological role of blood Syn is still elusive. Starting from the notion that platelets are either the major cellular reservoir of Syn in the blood and, concomitantly, act as key players in hemostasis, being activated also by -thrombin (T) via cleavage of protease-activated receptors (PARs), we decided to investigate the possibility that Syn could modulate platelet activation by interfering with the T-PAR functional axis. Using multiple electrode aggregometry, i.e. a fast and specific platelet-function-testing method, as well as steady-state fluorescence spectroscopy, surface plasmon resonance, and fluorescence microscopy, we show here that monomeric Syn functions as a negative regulator of T-mediated platelets activation. Syn acts either directly, via competitive inhibition of PAR1 activation by T and TRAP6 agonist, and indirectly, by scavenging T on the platelet plasma membrane. A simple electrostatic model of Syn platelet antiaggregating effect is proposed and the possible role of the protein at the interplay of amyloidosis and thrombosis is discussed.

L-fucokinase (FUK) and GDP-fucose pyrophosphorylase (GFPP) salvage free L-fucose and synthesize the valuable nucleotide-sugar GDP-L-fucose (GDP-Fuc). Some organisms express these enzymes as one bifunctional polypeptide called L-fucokinase/GDP-fucose pyrophosphorylase (FKP), which has attracted attention for use in the chemoenzymatic synthesis of GDP-Fuc. Despite the documented use of the FKP from Bacteroides fragilis (BfFKP), the evolutionary origins of these enzymes and their relationships to monofunctional FUKs and GFPPs are poorly understood. We hypothesized that biochemical characterization of these proteins coupled with an evolutionary analysis would uncover the natural diversity of FKPs, facilitating the discovery of new biocatalysts. Phylogenetic and sequence similarity network (SSN) analyses distinguished FKPs from FUKs and GFPPs, suggesting that FKPs originate from one ancestral fusion event between these domains. To evaluate how environmental factors might select for functional diversity within the FKP family, we recombinantly expressed and purified a putative FKP from the thermophilic bacterium Thermophagus xiamenensis (TxFKP). This enzyme exhibited in vitro kinase and pyrophosphorylase activities and demonstrated subtle kinetic differences compared to BfFKP. While alanine scanning mutational analysis of the TxFKP FUK and GFPP domains supported the role of conserved residues that TxFKP uses to coordinate substrate binding and catalysis, other mutations in the TxFKP GFPP domain influenced kinase activity differentially for the substrates L-fucose and D-arabinose, showing an unprecedented role for the GFPP domain in FUK substrate specificity. Finally, thermal shift profiles of TxFKP and BfFKP were biphasic and provided new insights into how these enzymes have evolved to respond to different sugar substrates.

TAR binding protein 43 (TDP-43) is an RNA binding protein that assists in the maturation, export and sub-cellular localization of mRNA. The carboxyl terminal 153 residues of TDP-43 are of low sequence complexity and allow for self-association of the protein in a manner leading to its phase separation from an aqueous environment. These interactions assist TDP-43 in forming cytoplasmic RNA granules involved in the transport of mRNA for localized translation. Self-association of the TDP-43 low complexity (LC) domain is facilitated by a region of twenty five residues that are of extreme evolutionary conservation. The molecular basis for self-adherence of the protein through this region has been illuminated by a combination of structural and biochemical studies, allowing definition of a morphologically specific cross-{beta} structure predicted to be weakly assembled by main chain hydrogen bonds. In this study we have investigated the importance of individual, Pauling hydrogen bonds hypothesized to facilitate self-adherence of the TDP-43 LC domain.