Exploring the significance of microbiota metabolites in rheumatoid arthritis: uncovering their contribution from disease development to biomarker potential.

Rheumatoid arthritis (RA) is a multifaceted autoimmune disorder characterized by chronic inflammation and joint destruction. Recent research has elucidated the intricate interplay between gut microbiota and RA pathogenesis, underscoring the role of microbiota-derived metabolites as pivotal contributors to disease development and progression. The human gut microbiota, comprising a vast array of microorganisms and their metabolic byproducts, plays a crucial role in maintaining immune homeostasis. Dysbiosis of this microbial community has been linked to numerous autoimmune disorders, including RA. Microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), tryptophan derivatives, Trimethylamine-N-oxide (TMAO), bile acids, peptidoglycan, and lipopolysaccharide (LPS), exhibit immunomodulatory properties that can either exacerbate or ameliorate inflammation in RA. Mechanistically, these metabolites influence immune cell differentiation, cytokine production, and gut barrier integrity, collectively shaping the autoimmune milieu. This review highlights recent advances in understanding the intricate crosstalk between microbiota metabolites and RA pathogenesis and also discusses the potential of specific metabolites to trigger or suppress autoimmunity, shedding light on their molecular interactions with immune cells and signaling pathways. Additionally, this review explores the translational aspects of microbiota metabolites as diagnostic and prognostic tools in RA. Furthermore, the challenges and prospects of translating these findings into clinical practice are critically examined.

PARP1's Involvement in RNA Polymerase II Elongation: Pausing and Releasing Regulation through the Integrator and Super Elongation Complex.

RNA polymerase elongation along the gene body is tightly regulated to ensure proper transcription and alternative splicing events. Understanding the mechanism and factors critical in regulating the rate of RNA polymerase II elongation and processivity is clearly important. Recently we showed that PARP1, a well-known DNA repair protein, when bound to chromatin, regulates RNA polymerase II elongation. However, the mechanism by which it does so is not known. In the current study, we aimed to tease out how PARP1 regulates RNAPII elongation. We show, both in vivo and in vitro, that PARP1 binds directly to the Integrator subunit 3 (IntS3), a member of the elongation Integrator complex. The association between the two proteins is mediated via the C-terminal domain of PARP1 to the C-terminal domain of IntS3. Interestingly, the occupancy of IntS3 along two PARP1 target genes mimicked that of PARP1, suggesting a role in its recruitment/assembly of elongation factors. Indeed, the knockdown of PARP1 resulted in differential chromatin association and gene occupancy of IntS3 and other key elongation factors. Most of these PARP1-mediated effects were due to the physical presence of PARP1 rather than its PARylation activity. These studies argue that PARP1 controls the progressive RNAPII elongation complexes. In summary, we present a platform to begin to decipher PARP1's role in recruiting/scaffolding elongation factors along the gene body regions during RNA polymerase II elongation and gene regulation.

Evidence Supporting Substrate Channeling between Domains of Human PAICS: A Time-Course Analysis of 13C-Bicarbonate Incorporation.

Human phosphoribosylaminoimidazole carboxylase phosphoribosylaminoimdiazole succinocarboxamide synthetase (PAICS) is a dual activity enzyme catalyzing two consecutive reactions in de novo purine nucleotide synthesis. Crystallographic structures of recombinant human PAICS suggested the channeling of 4-carboxy-5-aminoimidazole-1-ribose-5'-phosphate (CAIR) between two active sites of PAICS, while a prior work of an avian PAICS suggested otherwise. Here, we present time-course mass spectrometric data supporting the channeling of CAIR between domains of recombinant human PAICS. Time-course mass spectral analysis showed that CAIR added to the bulk solution (CAIRbulk) is decarboxylated and re-carboxylated before the accumulation of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR). An experiment with 13C-bicarbonate showed that SAICAR production was proportional to re-carboxylated CAIR instead of total CAIR or CAIRbulk. This result indicates that the SAICAR synthase domain selectively uses enzyme-made CAIR over CAIRbulk, which is consistent with the channeling model. This channeling between PAICS domains may be a part of a larger channeling process in de novo purine nucleotide synthesis.

PARP1 is a versatile factor in the regulation of mRNA stability and decay.

PARP1 is an abundant nuclear protein with many pleiotropic functions involved in epigenetic and transcriptional controls. Abundance of mRNA depends on the balance between synthesis and decay of a particular transcript. PARP1 binds RNA and its depletion results in increased expression of genes involved in nonsense-mediated decay, suggesting that PARP1 might be involved in mRNA stability. This is of interest considering RNA binding proteins play key roles in post-transcriptional processes in all eukaryotes. We tested the direct impact of PARP1 and PARylation on mRNA stability and decay. By measuring the half-lives of two PARP1-mRNA targets we found that the half-lives were significantly decreased in PARP1-depleted cells. PARP1 depletion impacted both the synthesis of nascent mRNA and the stability of mature mRNAs. PARylation impacted the production of nascent mRNA and the stability of mature mRNA, albeit to a lesser extent than PARP1 KD. PARylation enhanced the impact of PARP1 depletion. These studies provide the first direct comparative role of PARP1 and PARylation in RNA stability and decay, adding a new dimension as to how PARP1 regulates gene expression. These studies present a platform to begin to tease out the influence of PARP1 at each step of RNA biogenesis and decay to fine-tune gene expression.

Coupling of PARP1-mediated chromatin structural changes to transcriptional RNA polymerase II elongation and cotranscriptional splicing.

BACKGROUND: Recently, we showed that PARP1 is involved in cotranscriptional splicing, possibly by bridging chromatin to RNA and recruiting splicing factors. It also can influence alternative splicing decisions through the regulation of RNAPII elongation. In this study, we investigated the effect of PARP1-mediated chromatin changes on RNAPII movement, during transcription and alternative splicing. RESULTS: We show that RNAPII pauses at PARP1-chromatin structures within the gene body. Knockdown of PARP1 abolishes this RNAPII pausing, suggesting that PARP1 may regulate RNAPII elongation. Additionally, PARP1 alters nucleosome deposition and histone post-translational modifications at specific exon-intron boundaries, thereby affecting RNAPII movement. Lastly, genome-wide analyses confirmed that PARP1 influences changes in RNAPII elongation by either reducing or increasing the rate of RNAPII elongation depending on the chromatin context. CONCLUSIONS: These studies suggest a context-specific effect of PARP1-chromatin binding on RNA polymerase movement and provide a platform to delineate PARP1's role in RNA biogenesis and processing.

Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn-1 gene.

INTRODUCTION: In kindling, repeated electrical stimulation of certain brain areas causes progressive and permanent intensification of epileptiform activity resulting in generalized seizures. We focused on the role(s) of glutamate and a negative regulator of glutamate release, STXBP5/tomosyn-1, in kindling. METHODS: Stimulating electrodes were implanted in the amygdala and progression to two successive Racine stage 5 seizures was measured in wild-type and STXBP5/tomosyn-1-/- (Tom-/-) animals. Glutamate release measurements were performed in distinct brain regions using a glutamate-selective microelectrode array (MEA). RESULTS: Naïve Tom-/- mice had significant increases in KCl-evoked glutamate release compared to naïve wild type as measured by MEA of presynaptic release in the hippocampal dentate gyrus (DG). Kindling progression was considerably accelerated in Tom-/- mice, requiring fewer stimuli to reach a fully kindled state. Following full kindling, MEA measurements of both kindled Tom+/+ and Tom-/- mice showed significant increases in KCl-evoked and spontaneous glutamate release in the DG, indicating a correlation with the fully kindled state independent of genotype. Resting glutamate levels in all hippocampal subregions were significantly lower in the kindled Tom-/- mice, suggesting possible changes in basal control of glutamate circuitry in the kindled Tom-/- mice. CONCLUSIONS: Our studies demonstrate that increased glutamate release in the hippocampal DG correlates with acceleration of the kindling process. Although STXBP5/tomosyn-1 loss increased evoked glutamate release in naïve animals contributing to their prokindling phenotype, the kindling process can override any attenuating effect of STXBP5/tomosyn-1. Loss of this "braking" effect of STXBP5/tomosyn-1 on kindling progression may set in motion an alternative but ultimately equally ineffective compensatory response, detected here as reduced basal glutamate release.

Vimentin is involved in regulation of mitochondrial motility and membrane potential by Rac1.

In this study we show that binding of mitochondria to vimentin intermediate filaments (VIF) is regulated by GTPase Rac1. The activation of Rac1 leads to a redoubling of mitochondrial motility in murine fibroblasts. Using double-mutants Rac1(G12V, F37L) and Rac1(G12V, Y40H) that are capable to activate different effectors of Rac1, we show that mitochondrial movements are regulated through PAK1 kinase. The involvement of PAK1 kinase is also confirmed by the fact that expression of its auto inhibitory domain (PID) blocks the effect of activated Rac1 on mitochondrial motility. The observed effect of Rac1 and PAK1 kinase on mitochondria depends on phosphorylation of the Ser-55 of vimentin. Besides the effect on motility Rac1 activation also decreases the mitochondrial membrane potential (MMP) which is detected by ∼20% drop of the fluorescence intensity of mitochondria stained with the potential sensitive dye TMRM. One of important consequences of the discovered regulation of MMP by Rac1 and PAK1 is a spatial differentiation of mitochondria in polarized fibroblasts: at the front of the cell they are less energized (by ∼25%) than at the rear part.

Mitochondrial membrane potential is regulated by vimentin intermediate filaments.

This study demonstrates that the association of mitochondria with vimentin intermediate filaments (VIFs) measurably increases their membrane potential. This increase is detected by quantitatively comparing the fluorescence intensity of mitochondria stained with the membrane potential-sensitive dye tetramethylrhodamine-ethyl ester (TMRE) in murine vimentin-null fibroblasts with that in the same cells expressing human vimentin (∼35% rise). When vimentin expression is silenced by small hairpin RNA (shRNA) to reduce vimentin by 90%, the fluorescence intensity of mitochondria decreases by 20%. The increase in membrane potential is caused by specific interactions between a subdomain of the non-α-helical N terminus (residues 40 to 93) of vimentin and mitochondria. In rho 0 cells lacking mitochondrial DNA (mtDNA) and consequently missing several key proteins in the mitochondrial respiratory chain (ρ(0) cells), the membrane potential generated by an alternative anaerobic process is insensitive to the interactions between mitochondria and VIF. The results of our studies show that the close association between mitochondria and VIF is important both for determining their position in cells and their physiologic activity.

Kindling-induced asymmetric accumulation of hippocampal 7S SNARE complexes correlates with enhanced glutamate release.

PURPOSE: To correlate kindling-associated alterations of the neurotransmitter secretory machinery, glutamate release in the trisynaptic hippocampal excitatory pathway, and the behavioral evolution of kindling-induced epileptogenesis. METHOD: Neurotransmitter release requires the fusion of vesicle and plasma membranes; it is initiated by formation of a stable, ternary complex (7SC) of SNARE [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor] proteins. Quantitative Western blotting was used to monitor levels of 7SC and SNARE regulators [NSF, SV2 (synaptic vesicle protein 2)] in hippocampal synaptosomes from amygdala-kindled animals. Hippocampal synaptic glutamate release was measured in vivo with a unique microelectrode array (MEA) that uses glutamate oxidase to catalyze the breakdown of glutamate into a reporter molecule. KEY FINDINGS: Ipsilateral hippocampal accumulation of 7SC developed with onset of amygdalar kindling, but became permanent only in animals stimulated to at least Racine stage 3; the ratio peaked and did not increase with more than two consecutive stage 5 seizures. Chronic 7SC asymmetry was seen in entorhinal cortex and the hippocampal formation, particularly in dentate gyrus (DG) and CA1, but not in the other brain areas examined. There was a strong correlation between asymmetric 7SC accumulation and increased total hippocampal SV2. Following a 30-day latent period, amplitudes of spontaneous synaptic glutamate release were enhanced in ipsilateral DG and reduced in ipsilateral CA3 of kindled animals; increased volleys of synaptic glutamate activity were seen in ipsilateral CA1. SIGNIFICANCE: Amygdalar kindling is associated with chronic changes in the flow of glutamate signaling in the excitatory trisynaptic pathway and with early but permanent changes in the mechanics of vesicular release in ipsilateral hippocampal formation.

Dissecting the N-ethylmaleimide-sensitive factor: required elements of the N and D1 domains.

N-Ethylmaleimide-sensitive factor (NSF) is a homo-hexameric member of the AAA(+) (ATPases associated with various cellular activities plus) family. It plays an essential role in most intracellular membrane trafficking through its binding to and disassembly of soluble NSF attachment protein (SNAP) receptor (SNARE) complexes. Each NSF protomer contains an N-terminal domain (NSF-N) and two AAA domains, a catalytic NSF-D1 and a structural NSF-D2. This study presents detailed mutagenesis analyses of NSF-N and NSF-D1, dissecting their roles in ATP hydrolysis, SNAP.SNARE binding, and complex disassembly. Our results show that a positively charged surface on NSF-N, bounded by Arg(67) and Lys(105), and the conserved residues in the central pore of NSF-D1 (Tyr(296) and Gly(298)) are involved in SNAP.SNARE binding but not basal ATP hydrolysis. Mutagenesis of Sensor 1 (Thr(373)-Arg(375)), Sensor 2 (Glu(440)-Glu(442)), and Arginine Fingers (Arg(385) and Arg(388)) in NSF-D1 shows that each region plays a discrete role. Sensor 1 is important for basal ATPase activity and nucleotide binding. Sensor 2 plays a role in ATP- and SNAP-dependent SNARE complex binding and disassembly but does so in cis and not through inter-protomer interactions. Arginine Fingers are important for SNAP.SNARE complex-stimulated ATPase activity and complex disassembly. Mutants at these residues have a dominant-negative phenotype in cells, suggesting that Arginine Fingers function in trans via inter-protomer interactions. Taken together, these data establish functional roles for many of the structural elements of the N domain and of the D1 ATP-binding site of NSF.

Levetiracetam prevents kindling-induced asymmetric accumulation of hippocampal 7S SNARE complexes.

PURPOSE: Understanding the molecular mechanisms underlying epilepsy is crucial to designing novel therapeutic regimens. This report focuses on alterations in the secretory machinery responsible for neurotransmitter (NT) release. Soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) complexes mediate the fusion of synaptic vesicle and active zone membranes, thus mediating NT secretion. SNARE regulators control where and when SNARE complexes are formed. Previous studies showed an asymmetric accumulation of 7S SNARE complexes (7SC) in the ipsilateral hippocampus of kindled animals. The present studies probe the persistence of 7SC accumulation and the effect of the anticonvulsant, levetiracetam (LEV), on 7SC and SNARE regulators. METHOD: Quantitative Western blotting was used to monitor levels of 7SC and SNARE regulators in hippocampal synaptosomes from kindled animals both before and after LEV treatment. RESULTS: The asymmetric accumulation of 7SC is present 1-year postamygdalar kindling. The synaptic vesicle protein, synaptic vesicle protein 2 (SV2), a primary LEV-binding protein, and the SNARE regulator Tomosyn increase, whereas NSF decreases in association with this accumulation. Treatment with LEV prevented kindling-induced accumulation of SV2, but did not affect the transient increase of Tomosyn or the long-term decrease NSF. LEV treatment retarded the electrical and behavioral concomitants of amygdalar kindling coincident with a decrease in accumulation of 7SC. CONCLUSIONS: The ipsilateral hippocampal accumulation of SNARE complexes is an altered molecular process associated with kindling that appears permanent. Kindling epileptogenesis alters synaptosomal levels of the SNARE regulators: NSF, SV2, and Tomosyn. Concomitant treatment with LEV reverses the kindling-induced 7SC accumulation and increase of SV2.

Asymmetric accumulation of hippocampal 7S SNARE complexes occurs regardless of kindling paradigm.

Modifications of neurotransmission may contribute to the synchronization of neuronal networks that are a hallmark of epileptic seizures. In this study we examine the synaptosomal proteins involved in neurotransmitter release to determine if alterations in their interactions correlate with the chronic epileptic state. Using quantitative western blotting, we measured the levels of 7S SNARE complexes and SNARE effectors in the effected hippocampi from animals that were electrically kindled through stimulation from one of three different foci. All three kindling paradigms, amygdalar, entorhinal, and septal, were associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus, measured 1 month after completion of kindling. Of the eight SNARE effectors examined (alpha-SNAP, NSF, SV2A/B, Munc18a/nSec1, Munc13-1, Complexins 1 and 2, and synaptotagmin I), there was a statistically significant bihemispheric increase of hippocampal SV2 and decrease of NSF upon kindling; neither by itself would be expected to account for the asymmetry of SNARE complex distribution. These data suggest that an ipsilateral hippocampal accumulation of SNARE complexes is a permanent alteration of kindling-induced epilepsy, regardless of stimulation pathway. The significance of these findings toward a molecular understanding of epilepsy will be discussed.

Type I PDZ ligands are sufficient to promote rapid recycling of G Protein-coupled receptors independent of binding to N-ethylmaleimide-sensitive factor.

Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the beta2 adrenergic receptor (beta2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the beta2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the beta2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the beta1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the beta2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.

Multiple binding proteins suggest diverse functions for the N-ethylmaleimide sensitive factor.

The hexameric ATPase, N-ethylmaleimide sensitive factor (NSF), is essential to vesicular transport and membrane fusion because it affects the conformations and associations of the soluble NSF attachment protein receptor (SNARE) proteins. NSF binds SNAREs through adaptors called soluble NSF attachment proteins (alpha- or beta-SNAP) and disassembles SNARE complexes to recycle the monomers. NSF contains three domains, two nucleotide-binding domains (NSF-D1 and -D2) and an amino terminal domain (NSF-N) that is required for SNAP-SNARE complex binding. Mutagenesis studies indicate that a cleft between the two sub-domains of NSF-N is critical for binding. The structural conservation of N domains in NSF, p97/VCP, and VAT suggests that a similar type of binding site could mediate substrate recognition by other AAA proteins. In addition to SNAP-SNARE complexes, NSF also binds other proteins and protein complexes such as AMPA receptor subunits (GluR2), beta2-adrenergic receptor, beta-Arrestin1, GATE-16, LMA1, rabs, and rab-containing complexes. The potential for these interactions indicates a broader role for NSF in the assembly/disassembly cycles of several cellular complexes and suggests that NSF may have specific regulatory effects on the functions of the proteins involved in these complexes. The structural requirements for these interactions and their physiological significance will be discussed.

Uncoupling the ATPase activity of the N-ethylmaleimide sensitive factor (NSF) from 20S complex disassembly.

The N-ethylmaleimide sensitive factor (NSF) plays a critical role in intracellular trafficking by disassembling soluble NSF attachment protein receptor (SNARE ) complexes. The NSF protomer consists of three domains (NSF-N, NSF-D1, and NSF-D2). Two domains (NSF-D1 and NSF-D2) contain a conserved approximately 230 amino acid cassette, which includes a distinctive motif termed the second region of homology (SRH) common to all ATPases associated with various cellular activities (AAA). In hexameric NSF, several SRH residues become trans elements of the ATP binding pocket. Mutation of two conserved arginine residues in the NSF-D1 SRH (R385A and R388A) did not effect basal or soluble NSF attachment protein (SNAP)-stimulated ATPase activity; however, neither mutant underwent ATP-dependent release from SNAP-SNARE complexes. A trans element of the NSF-D2 ATP binding site (K631) has been proposed to limit the ATPase activity of NSF-D2, but a K631D mutant retained wild-type activity. A mutation of the equivalent residue in NSF-D1 (D359K) also did not affect nucleotide hydrolysis activity but did limit NSF release from SNAP-SNARE complexes. These trans elements of the NSF-D1 ATP binding site (R385, R388, and D359) are not required for nucleotide hydrolysis but are important as nucleotide-state sensors. NSF-N mediates binding to the SNAP-SNARE complex. To identify the structural features required for binding, three conserved residues (R67, S73, and Q76) on the surface of NSF-N were mutated. R67E completely eliminated binding, while S73R and Q76E showed limited effect. This suggests that the surface important for SNAP binding site lies in the cleft between the NSF-N subdomains adjacent to a conserved, positively charged surface.