Regulation Of Transport Proteins Research Articles

UBL3 Interacts with PolyQ-Expanded Huntingtin Fragments and Modifies Their Intracellular Sorting.

UBL3 (Ubiquitin-like 3) is a protein that plays a crucial role in post-translational modifications, particularly in regulating protein transport within small extracellular vesicles. While previous research has predominantly focused on its interactions with α-synuclein, this study investigates UBL3's role in Huntington's disease (HD). HD is characterized by movement disorders and cognitive impairments, with its pathogenesis linked to toxic, polyglutamine (polyQ)-expanded mutant huntingtin fragments (mHTT). However, the mechanisms underlying the interaction between UBL3 and mHTT remain poorly understood. To elucidate this relationship, we performed hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) on postmortem brain tissue from HD patients. Gaussia princeps-based split-luciferase complementation assay and co-immunoprecipitation were employed to confirm the interaction between UBL3 and mHTT. Additionally, we conducted a HiBiT lytic detection assay to assess the influence of UBL3 on the intracellular sorting of mHTT. Finally, immunocytochemical staining was utilized to validate the colocalization and distribution of these proteins. Our findings revealed UBL3-positive inclusions in the cytoplasm and nuclei of neurons throughout the striatum of HD patients. We discovered that UBL3 colocalizes and interacts with mHTT and modulates its intracellular sorting. These results suggest that UBL3 may play a significant role in the interaction and sorting of mHTT, contributing to the understanding of its potential implications in the pathophysiology of Huntington's disease.

Spatiotemporal patterns of locus coeruleus integrity predict cortical tau and cognition.

Autopsy studies indicated that the locus coeruleus (LC) accumulates hyperphosphorylated tau before allocortical regions in Alzheimer's disease. By combining in vivo longitudinal magnetic resonance imaging measures of LC integrity, tau positron emission tomography imaging and cognition with autopsy data and transcriptomic information, we examined whether LC changes precede allocortical tau deposition and whether specific genetic features underlie LC's selective vulnerability to tau. We found that LC integrity changes preceded medial temporal lobe tau accumulation, and together these processes were associated with lower cognitive performance. Common gene expression profiles between LC-medial temporal lobe-limbic regions map to biological functions in protein transport regulation. These findings advance our understanding of the spatiotemporal patterns of initial tau spreading from the LC and LC's selective vulnerability to Alzheimer's disease pathology. LC integrity measures can be a promising indicator for identifying the time window when individuals are at risk of disease progression and underscore the importance of interventions mitigating initial tau spread.

Differential analysis of ubiquitin-proteomics in skeletal muscle of Duroc pigs and Tibetan fragrant pigs.

Understanding the differences in ubiquitination-modified proteins between Duroc pigs and Tibetan fragrant pigs is crucial for comprehending the growth and development of their skeletal muscles. In this study, skeletal muscle samples from 30-day-old Duroc pigs and Tibetan fragrant pigs were collected. Using ubiquitination 4D-Label free quantitative proteomics, we analyzed and identified ubiquitination-modified peptides, screening out 109 differentially expressed ubiquitination-modified peptides. Further enrichment analysis was conducted on the proteins associated with these differential peptides. GO analysis results indicated that the differential genes were primarily enriched in processes such as regulation of protein transport, motor activity, myosin complex, and actin cytoskeleton. KEGG pathway analysis revealed significant enrichment in pathways such as Glycolysis/Gluconeogenesis and Hippo signaling pathway. The differentially expressed key ubiquitinated proteins, including MYL1, MYH3, TNNC2, TNNI1, MYLPF, MYH1, MYH7, TNNT2, TTN, and TNNC1, were further identified. Our analysis demonstrates that these genes play significant roles in skeletal muscle protein synthesis and degradation, providing new insights into the molecular mechanisms of muscle development in Duroc pigs and Tibetan fragrant pigs, and offering theoretical support for breeding improvements in the swine industry.

Spatiotemporal Assessment of Locus Coeruleus Integrity Predicting Cortical Tau and Cognition

AbstractBackgroundMisfolded tau protein, an Alzheimer’s Disease (AD) hallmark, accumulates decades before the emergence of cognitive decline. Autopsy and neuroimaging studies support the locus coeruleus (LC) as an early site of tau and its contribution to disease progression. However, whether tau in LC precedes cortical tau deposition remains unclear. Understanding the topography of tau progression and the biological factors making specific neuronal systems prone to AD‐related pathology is essential to target interventions appropriately. We examined the spatiotemporal relationships between LC integrity and cortical tau accumulation and its relevance to cognition.,MethodsWe combined longitudinal LC‐integrity (T1‐TSE‐imaging) and tau pathology (18F‐FTP‐PET) data from 77 adults (Fig1A). We used whole‐brain voxel‐wise GLM analysis to investigate the relationship between baseline LC‐integrity (inverted signal) and longitudinal tau accumulation and compared the correlation distributions in both directions. Robust regression analysis was used to examine whether the observed tau spreading pathway predicts PACC5 performance at follow‐up. We used neuropathological measures from 160 cognitively unimpaired or MCI/AD adults (Fig1B) to support the in‐vivo neuroimaging data. Spearman‐rank partial correlations were used to relate LC tangle density to tangles in temporal cortex areas. Using AHBA, the biological backgrounds underlying the connectomic‐genetic relationships related to LC were explored using a whole‐brain region‐wise transcriptomic similarity analysis.,ResultsCorrelations between baseline LC‐integrity and follow‐up tau were stronger than the inverse correlations, indicative of lower LC‐integrity preceding tau accumulation in MTL (Fig1C‐D). LC tangle density was strongly related to tangles in MTL structures (Fig1E). Longitudinal tau accumulation in the LC‐MTL axis is associated with lower cognitive performance (Fig2). Common neurogenetic profiles exist between LC and MTL/limbic regions, supporting a shared connectomic‐transcriptomic substrate. The genetic profile displays specific biological functions in protein transport regulation and lipid biosynthetic processes (Fig3).,ConclusionOur results suggest that changes in LC‐integrity may occur before tau spreads into the MTL, which jointly contributes to lower cognitive performance. The neurogenetic profiles can provide a biological framework for identifying individuals more likely to be at risk for AD disease progression. Future developments could examine the intersection between amyloid and LC‐related tau progression in the context of AD.

XPO2 Is Essential for a Subset of AML Cells and Is a Biomarker for Poor Response and Survival in Pediatric and AYA AML

Importins & exportins regulate protein transport across the nuclear membrane. Twenty nuclear transport receptors (NTRs) have been identified; six are involved in nuclear export. NTRs contribute to cancer development, through abnormal localization of protein cargos. The exportin, XPO1, is associated with high risk AML & shorter survival times. However, little is known regarding the functional importance of the related exportin, XPO2 (alias CSE1L). We analyzed a public database of CRISPR/Cas9 depletion screens in 26 AML cell lines. XPO2 ranked in the top 1.4% of essential genes, along with XPO1 (top 1.3%) (DepMap 22Q2 Public+Score, Chronos). An RNAi depletion screen confirmed this, with XPO2 ranked in the top 0.2% of AML genes. Leukemia cells were more dependent on XPO2 vs solid tumors (P = 0.0394) & gene dependency scores indicated a greater reliance on XPO2 vs XPO1 (P <0.0001). Increased expression of XPO2 mRNA was associated with decreased event free survival (EFS) (P = 0.0014) & overall survival (OS) (P = 0.00091) in pediatric & adolescent/young adult (AYA) AML patients (TARGET dataset). In the BeatAML dataset, increased XPO2 mRNA expression was associated with decreased OS in patients <40 years, but not in patients >40 years (P <0.0001). We analyzed protein expression databases & identified XPO2 as the highest expressed exportin in AML cells lines & patient samples. Increased XPO2 expression was detected in 7/7 primary AML samples & 9 AML cell lines by immunoblot (HL60, MV4-11, MOML13, NB4, OCI-AML2, OCI-M2, TEX, U937 & 1254 cells), compared to normal hematopoietic cells. Expression of XPO2 was increased in younger (<40 years) vs older patients (>70 years) (P = 0.0454). Genetic silencing of XPO2 decreased growth & viability of OCI-AML2, TEX, NB4 & 1254 cells. Gambogic acid is an inhibitor of XPO2-mediated nuclear transport. In OCI-AML2, TEX & NB4 cell lines, gambogic acid decreased viability of AML cells (TEX IC50 = 369nM, OCI-AML2 IC50 = 433nM, NB4 IC50 = 161nM). Next, we interrogated a BioID database (PXD007976) & performed gene set enrichment analysis on XPO2 targets. Processes related to ribosome biogenesis were enriched in XPO2 cargo, whereas XPO1 cargo were enriched for snRNA transcription processes (P <0.05). A gene expression signature encompassing XPO2 & its associated cargo genes was enriched in functionally defined LSCs (P = 0.001, in patients with more primitive AML subtypes (P < 2.22 x 10 -16), in more primitive AML blasts as defined by FAB subtype and in high risk subsets, including WT1 mutated pediatric AML. Additionally, the XPO2 gene signature was associated with minimal-residual-disease (MRD) positive disease after induction cycle I (P < 5.1x 10 -6) and II (P = 0.0013), decreased complete remission (CR) rates after cycle I (P = 0.00025) and II (P = 0.02), worse EFS (P = 0.019) and worse OS (P = 0.02) in pediatric and AYA AML patients. In summary, we identified XPO2 as an essential gene for AML . XPO2 is increased in AML cells & primary samples from younger patients. Increased XPO2 expression & increased expression of XPO2 mediated processesare associated with worse outcomes in pediatric AML & correlate with LSC enrichment & primitive AML subtypes. XPO2 may function to enhance ribosomal biogenesis, demonstrating a critical role in leukemogenesis. Thus, this work demonstrates a new dependency of pediatric AML on XPO2 highlighting a new target and biomarker for this disease.

RhoGAP6 interacts with COPI to regulate protein transport.

RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets.

Blood-brain barrier integrity impacts the use of plasma amyloid-β as a proxy of brain amyloid-β pathology.

Amyloid-β (Aβ) and tau can be quantified in blood. However, biological factors can influence the levels of brain-derived proteins in the blood. The blood-brain barrier (BBB) regulates protein transport between cerebrospinal fluid (CSF) and blood. BBB altered permeability might affect the relationship between brain and blood biomarkers. We assessed 224 participants in research (TRIAD, n=96) and clinical (BIODEGMAR, n=128) cohorts with plasma and CSF/positron emission tomography Aβ, p-tau, and albumin measures. Plasma Aβ42/40 better identified CSF Aβ42/40 and Aβ-PET positivity in individuals with high BBB permeability. An interaction between plasma Aβ42/40 and BBB permeability on CSF Aβ42/40 was observed. Voxel-wise models estimated that the association of positron emission tomography (PET), with plasma Aβ was most affected by BBB permeability in AD-related brain regions. BBB permeability did not significantly impact the relationship between brain and plasma p-tau levels. These findings suggest that BBB integrity may influence the performance of plasma Aβ, but not p-tau, biomarkers in research and clinical settings. BBB permeability affects the association between brain and plasma Aβ levels. BBB integrity does not affect the association between brain and plasma p-tau levels. Plasma Aβ was most affected by BBB permeability in AD-related brain regions. BBB permeability increases with age but not according to cognitive status.

The bacterial secretosome for bacterial envelope biogenesis.

The Gram-negative bacterial envelope is a robust line of defence against physical and chemical environmental stress, including antibiotics. Therefore, its biogenesis is of considerable interest, as well as a target to address the growing problem of antimicrobial resistance. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machineries. The Sec translocon, a ubiquitous integral inner-membrane complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed into the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm, or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core hetero-trimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein transport and quality control. My talk will overview recent work on the association of the translocons of the inner- (Sec) and outer-membrane (BAM), along with periplasmic chaperones for the formation of the bacterial secretosome. I will describe our findings on the structure, dynamics and orchestration of this inter-membrane assembly for efficient passage of proteins into the inner-membrane, periplasm and outer-membrane - critical for efficient envelope biogenesis.

Fabrication of Redox-Controllable Bioinspired Nanochannels for Precisely Regulating Protein Transport.

Redox regulation is an inherent feature of nature and plays a crucial role in the transport of ions/small molecules. However, whether redox status affects the biomolecule transport remains largely unknown. To explore the effects of redox status on biomolecule transport, herein, we constructed a glutathione/glutathione disulfide (GSH/GSSG)-driven and pillar[5]arene (P5)-modified artificial nanochannel for protein transport. The results indicate that hemoglobin (Hb) protein is selectively and effectively transported across the GSH-driven P5-modified nanochannel, which suggests that the redox status of the nanochannel could affect the process of protein transport. Therefore, this redox-driven nanochannel could provide a potential application for biomolecule detection and redox-controllable biomolecular drug release.

In silico identification of miRNAs potentially targeting the genes that regulate protein transport and translocation in cattle in heat stress response

MicroRNAs (miRNAs) are involved in post-transcriptional gene regulation and single miRNAs regulating multiple mRNAs targets. Using bioinformatics approaches, this work aims to identify and characterize the miRNAs that could potentially bind with PDKCC, AP3S2 and SEC61A2 gene which regulate protein transport during heat stress. To resolve this problem, three freely available online miRNAs target finder databases were applied to identify the potential miRNAs which may target PDKCC, AP3S2 and SEC61A2 gene. The prediction results showed that a total of 115, 67 and 25 miRNAs for PDKCC, 267, 111 and 12 miRNAs for AP3S2 and 92, 120 and 31 miRNAs for SEC61A2 were identified as a possible potential candidate by TargetScan, MirTarget2 and miRanda respectively. Following the clustering process, we found eight miRNAs for PDKCC, three for AP3S2 and nine for SEC61A2s identified as possible candidates by all three databases. Five miRNAs in PDKCC, four in AP3S2 and five in the SEC61A2 gene have 100% identical sequence resemblance between human and bovine miRNAs. miRNAs binding site analysis showed that miR-34a in AP3S2 and miR-377 in SEC61A2 has the highest number of binding sites while miR-30b and miR-30c have two binding sites in 3´UTR of SEC61A2 gene whereas the rest of the miRNAs have the lowest number of the binding site in PDKCC and SEC61A2 gene respectively. Our study identified total four potential miRNAs that have different binding sites at the AP3S2 and SEC61A2 3´UTR site along with seven miRNAs which have single binding site and can modulate protein transport, translocation and other cellular pathways.

Ultraviolet/Visible Light Regulated Protein Transport Gate Constructed by Pillar[6]arene-based Host-Guest System.

Protein transport is an interesting and intrinsic life feature that is highly relevant to physiology and disease in living beings. Herein, inspired by nature, based on the supramolecular host-guest interaction, we have introduced the classical azobenzene light switches and L-phenylalanine derived pillar[6]arene (L-Phe-P6) into the artificial nanochannel to construct light-responsive nanochannels that could regulate protein transport effectively under the control of ultraviolet (UV) and visible (Vis) light. The light-controlled distribution of L-Phe-P6 in the channel led to the difference in surface charges in the nanochannel, which eventually brought the difference in protein transport. This research may not only provide a convenient theoretical model for biological research, but also a flexible light-responsive protein transport model, which will play a crucial role in light-controlled release of protein drugs and so on.

Evaluating the regulation of transporter proteins and P-glycoprotein in rats with cholestasis and its implication for digoxin clearance.

BACKGROUNDCardiac and hepatic functionality are intertwined in a multifaceted relationship. Pathologic processes involving one may affect the other through a variety of mechanisms, including hemodynamic and membrane transport effects.AIMTo better understand the effect of extrahepatic cholestasis on regulations of membrane transporters involving digoxin and its implication for digoxin clearance.METHODSTwelve adult rats were included in this study; baseline hepatic and renal laboratory values and digoxin pharmacokinetic (PK) studies were established before evenly dividing them into two groups to undergo bile duct ligation (BDL) or a sham procedure. After 7 d repeat digoxin PK studies were completed and tissue samples were taken to determine the expressions of cell membrane transport proteins by quantitative western blot and real-time polymerase chain reaction. Data were analyzed using SigmaStat 3.5. Means between pre-surgery and post-surgery in the same experimental group were compared by paired t-test, while independent t-test was employed to compare the means between sham and BDL groups.RESULTSDigoxin clearance was decreased and liver function, but not renal function, was impaired in BDL rats. BDL resulted in significant up-regulation of multidrug resistance 1 expression in the liver and kidney and its down-regulation in the small intestine. Organic anion transporting polypeptides (OATP)1A4 was up-regulated in the liver but down-regulated in intestine after BDL. OATP4C1 expression was markedly increased in the kidney following BDL.CONCLUSIONThe results suggest that cell membrane transporters of digoxin are regulated during extrahepatic cholestasis. These regulations are favorable for increasing digoxin excretion in the kidney and decreasing its absorption from the intestine to compensate for reduced digoxin clearance due to cholestasis.

ADAPTOR PROTEIN-1 complex-mediated post-Golgi trafficking is critical for pollen wall development in Arabidopsis.

Summary Primexine deposition is essential for the formation of pollen wall patterns and is precisely regulated by the tapetum and microspores. While tapetum‐ and/or microspore‐localized proteins are required for primexine biosynthesis, how their trafficking is established and controlled is poorly understood.In Arabidopsis thaliana, AP1σ1 and AP1σ2, two genes encoding the σ subunit of the trans‐Golgi network/early endosome (TGN/EE)‐localized ADAPTOR PROTEIN‐1 complex (AP‐1), are partially redundant for plant viability, and the loss of AP1σ1 function reduces male fertility due to defective primexine formation. Here, we investigated the role of AP‐1 in pollen wall formation.The deposition of Acyl‐CoA SYNTHETASE5 (ACOS5) and type III LIPID TRANSFER PROTEINs (LTPs) secreted from the anther tapetum, which are involved in exine formation, were impaired in ap1σ1 mutants. In addition, the microspore plasma membrane (PM) protein RUPTURED POLLEN GRAIN1 (RPG1), which regulates primexine deposition, accumulated abnormally at the TGN/EE in ap1σ1 mutants. We show that AP‐1μ recognizes the YXXΦ motif of RPG1, thereby regulating its PM abundance through endocytic trafficking, and that loss of AP1σ1 decreases the levels of other AP‐1 subunits at the TGN/EE.Our observations show that AP‐1‐mediated post‐Golgi trafficking plays a vital role in pollen wall development by regulating protein transport in tapetal cells and microspores.

Posttranslational regulation of transporters important for symbiotic interactions.

Coordinated sharing of nutritional resources is a central feature of symbiotic interactions, and, despite the importance of this topic, many questions remain concerning the identification, activity, and regulation of transporter proteins involved. Recent progress in obtaining genome and transcriptome sequences for symbiotic organisms provides a wealth of information on plant, fungal, and bacterial transporters that can be applied to these questions. In this update, we focus on legume-rhizobia and mycorrhizal symbioses and how transporters at the symbiotic interfaces can be regulated at the protein level. We point out areas where more research is needed and ways that an understanding of transporter mechanism and energetics can focus hypotheses. Protein phosphorylation is a predominant mechanism of posttranslational regulation of transporters in general and at the symbiotic interface specifically. Other mechanisms of transporter regulation, such as protein-protein interaction, including transporter multimerization, polar localization, and regulation by pH and membrane potential are also important at the symbiotic interface. Most of the transporters that function in the symbiotic interface are members of transporter families; we bring in relevant information on posttranslational regulation within transporter families to help generate hypotheses for transporter regulation at the symbiotic interface.

ITRAQ-based proteomics of testicular interstitial fluid during aging in mice

Advancing age is associated with a decline in fertility and testicular function in males. The testicular interstitial fluid (TIF) bathing the seminiferous tubules and testicular interstitium is considered an important part of the testicular microenvironment. However, the TIF proteome in mice and whether it changes with age remain unclear. This study aimed to map the TIF proteome and identify differentially abundant proteins (DEPs) among young, middle-aged, and old mice using isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry. A total of 1477 proteins were identified in murine TIF. The abundance of 706 proteins showed a linear change trend with age, of which 360 and 346 proteins increased and decreased, respectively. In addition, 45 age-related DEPs were identified (P < 0.05, with at least 1.2-fold up- or downregulation). Bioinformatic analyses revealed that these proteins were involved in “actin cytoskeleton organization,” “intrinsic apoptotic signaling pathway,” and “regulation of protein transport.” Comparative analysis with relevant proteomes previously reported further revealed the characteristics of mouse TIF proteome. Moreover, two of the age-related DEPs (Fga and Qsox1) were also found to be age-related differentially expressed proteins in human blood plasma and senescence-related secretome of human peritubular myoid cells. Taken together, these findings may represent the foundation for a better understanding of the molecular function of TIF and testicular aging.

MALDI-TOF mass spectrometric protein profiling of THP-1 cells and their microvesicles

Extracellular vesicles that are shed from the plasma membranes take an active part in intercellular communication, transporting a wide range of molecules, including proteins, lipids, nucleic acids and carbohydrates, being of great functional importance. One of the steps to better understanding of distant communications of cells and their regulatory mechanisms is a proteomic study of various extracellular vesicles, including microvesicles and exosomes. Pro-inflammatory cytokines produced by monocytes and individual complement system components play a key role in their specific functioning. The aim of this work was to study proteomic composition of THP-1 monocyte-like cells and their microvesicles. The MALDI-mass spectrometric analysis of electrophoretic protein fractions of cell lysates and microvesicles allowed for identifying 107 proteins that perform various functions. Among 19 determined functional groups, the largest ones comprise transcription regulators and proteins with unknown functions. The smallest functional groups include regulators of cell differentiation and development, proteins participating in immune response and inflammation, cellular receptors and their regulators, transporter and transport regulatory proteins, as well as cell proteins mediating adhesion and matrix structures, processing regulators, proteins of ubiquitin-proteasome system, intracellular signaling, autophagy and exocytosis regulators, chromatin structural proteins, hemostatic regulators, and peptide hormones. An intermediate position is occupied by cytokines and growth factors, enzymes, cytoskeleton and motor proteins, as well as RNA processing and translation regulators. The subsequent DAVID Functional Annotation Clustering analysis allowed for identifying the most common groups distributed by their molecular function, biological processes, and cellular component. Separately, in the microvesicles derived from THP-1 monocyte-like cells, proteins of the immune response and inflammation, cytokines and growth factors, intracellular signaling proteins, cell differentiation regulators and developmental proteins, as well as cell adhesion and matrix proteins were identified among other protein molecules. The data obtained on the partial proteome of THP-1 monocyte-like cells and their microvesicles extend the existing knowledge on distant communications between the cells and suggest new mechanisms of interaction between monocytes/macrophages and their microenvironment.

The role of UhpA in regulating the virulence gene expression in Edwardsiella piscicida.

Edwardsiella piscicida (E.piscicida) is an important fish pathogen. However, the mechanism of Glu6P transport regulatory protein UhpA how to affect the virulence gene expression in E.piscicida is still unclear. The results in this study showed that the metabolism-related gene expression of cysteine synthase (orf 1134) and sulphate transporter (ychM) in the uhpA mutant strain ΔuhpA was 0.76-fold and 0.68-fold lower than the ones in the wild strains (p<.05). The gene expression of ethA and ethB in the ΔuhpA strain was 0.80-fold and 0.72-fold lower than the ones in the wild strains (p<.05). However, the gene expression of fliC and flgN in the ΔuhpA was 1.51-fold and 1.21-fold higher than the ones in the wild strains (p<.05). The gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the ΔuhpA was 1.27-fold, 1.13-fold, 1.28-fold and 1.23-fold higher than the ones in the wild strains (p<.05). This suggested that the uhpA gene could regulate the key virulence gene expression, and the uhpA gene was associated with the pathogenicity of E.piscicida in fish.

DDHD1, but Not DDHD2, Suppresses Neurite Outgrowth in SH-SY5Y and PC12 Cells by Regulating Protein Transport From Recycling Endosomes.

DDHD1 and DDHD2 are both intracellular phospholipases A1 and hydrolyze phosphatidic acid in vitro. Given that phosphatidic acid participates in neurite outgrowth, we examined whether DDHD1 and DDHD2 regulate neurite outgrowth. Depletion of DDHD1 from SH-SY5Y and PC12 cells caused elongation of neurites, whereas DDHD2 depletion prevented neurite elongation. Rescue experiments demonstrated that the enzymatic activity of DDHD1 is necessary for the prevention of neurite elongation. Depletion of DDHD1 caused enlargement of early endosomes and stimulated tubulation of recycling endosomes positive for phosphatidic acid-binding proteins syndapin2 and MICAL-L1. Knockout of DDHD1 enhanced transferrin recycling from recycling endosomes to the cell surface. Our results suggest that DDHD1 negatively controls the formation of a local phosphatidic acid-rich domain in recycling endosomes that serves as a membrane source for neurite outgrowth.

Hspa13 Promotes Plasma Cell Production and Antibody Secretion.

The generation of large numbers of plasma cells (PCs) is a main factor in systemic lupus erythematosus (SLE). We hypothesize that Hspa13, a member of the heat shock protein family, plays a critical role in the control of PC differentiation. To test the hypothesis, we used lipopolysaccharide (LPS)-activated B cells and a newly established mouse line with a CD19cre-mediated, B cell–specific deletion of Hspa13: Hspa13 cKO mice. We found that Hspa13 mRNA was increased in PCs from atacicept-treated lupus-prone mice and in LPS-stimulated plasmablasts (PBs) and PCs. A critical finding was that PBs and PCs [but not naïve B cells and germinal center (GC) B cells] expressed high levels of Hspa13. In contrast, the Hspa13 cKO mice had a reduction in BPs, PCs, and antibodies induced in vitro by LPS and in vivo by sheep red blood cells (SRCs)- or 4-hydroxy-3-nitrophenylacetyl (NP)-immunization. Accordingly, the Hspa13 cKO mice had reduced class-switched and somatically hypermutated antibodies with defective affinity maturation. Our work also showed that Hspa13 interacts with proteins (e.g., Bcap31) in the endoplasmic reticulum (ER) to positively regulate protein transport from the ER to the cytosol. Importantly, Hspa13 mRNA was increased in B220+ cells from patients with multiple myeloma (MM) or SLE, whereas Hspa13 cKO led to reduced autoantibodies and proteinuria in both pristane-induced lupus and lupus-prone MRL/lpr mouse models. Collectively, our data suggest that Hspa13 is critical for PC development and may be a new target for eliminating pathologic PCs.

GRASP55 Is Dispensable for Normal Hematopoiesis but Necessary for Myc-Dependent Leukemic Growth.

Grasp55 is a ubiquitous Golgi stacking protein involved in autophagy, protein trafficking, and glucose deprivation sensing. The function of Grasp55 in protein trafficking has been attributed to its PDZ-mediated interaction with the C-terminal PDZ-binding motifs of protein cargos. We have recently shown that such an interaction occurs between Grasp55 and the adhesion molecule Jam-C, which plays a central role in stemness maintenance of hematopoietic and spermatogenic cells. Accordingly, we have found that Grasp55-deficient mice suffer from spermatogenesis defects similar to Jam-C knockout mice. However, whether Grasp55 is involved in the maintenance of immunohematopoietic homeostasis through regulation of protein transport and Jam-C expression remains unknown. In this study, we show that Grasp55 deficiency does not affect hematopoietic stem cell differentiation, engraftment, or mobilization, which are known to depend on expression of Grasp55-dependent protein cargos. In contrast, using an Myc-dependent leukemic model addicted to autophagy, we show that knockdown of Grasp55 in leukemic cells reduces spleen and bone marrow tumor burden upon i.v. leukemic engraftment. This is not due to reduced homing of Grasp55-deficient cells to these organs but to increased spontaneous apoptosis of Grasp55-deficient leukemic cells correlated with increased sensitivity of the cells to glucose deprivation. These results show that Grasp55 plays a role in Myc-transformed hematopoietic cells but not in normal hematopoietic cells in vivo.