Fusokines are chimeric proteins generated by the physical coupling of cytokines in a single polypeptide, resulting in proteins with highly pleiotropic activity and the potential to treat cancer and autoimmune ailments. For instance, the fusokine GIFT15 (GM-CSF and Interleukin 15 Fusion Transgene) has been shown to be a powerful immunosuppressive protein able to convert naì ve B cells into IL-10-producing B cells. To date, the mammalian cell systems used for the expression of GIFT15 allow for secretion of the protein in the culturing media, an inefficient system for producing GMPcompliant fusokines. In this study we report the bacterial expression of bioactive recombinant GIFT15 (rGIFT15). Indeed, there is a constant demand to improve the expression systems for therapeutic proteins. Expression of a maltose-binding protein (MBP) fusion protein efficiently allowed the accumulation of soluble protein in the intracellular milieu. Optimizing the bacterial culture significantly increased the yield of recombinant protein. The biological activity of rGIFT15 was comparable to that of fusokine derived from a mammalian source. This approach led to the production of soluble, endotoxin-free functional protein, averaging 5 mg of rGIFT15 per liter of culture. This process is amenable to scale up for the development of Food and Drug Administration (FDA)-compliant immune-modulatory rGIFT15.
The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.
The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin-mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.
Background
We recently identified U1 small nuclear ribonucleoprotein (snRNP) tangle-like aggregates and RNA splicing abnormalities in sporadic Alzheimer’s disease (AD). However little is known about snRNP biology in early onset AD due to autosomal dominant genetic mutations or trisomy 21 in Down syndrome. Therefore we investigated snRNP biochemical and pathologic features in these disorders.
Findings
We performed quantitative proteomics and immunohistochemistry in postmortem brain from genetic AD cases. Electron microscopy was used to characterize ultrastructural features of pathologic aggregates. U1-70k and other snRNPs were biochemically enriched in the insoluble fraction of human brain from subjects with presenilin 1 (PS1) mutations. Aggregates of U1 snRNP-immunoreactivity formed cytoplasmic tangle-like structures in cortex of AD subjects with PS1 and amyloid precursor protein (APP) mutations as well as trisomy 21. Ultrastructural analysis with electron microscopy in an APP mutation case demonstrated snRNP immunogold labeling of paired helical filaments (PHF).
Conclusions
These studies identify U1 snRNP pathologic changes in brain of early onset genetic forms of AD. Since dominant genetic mutations and trisomy 21 result in dysfunctional amyloid processing, the findings suggest that aberrant β-amyloid processing may influence U1 snRNP aggregate formation.
Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.
Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation and triggers neurodegeneration. AEP is upregulated and active during aging and is activated in human AD brain and tau P301S-transgenic mice with synaptic pathology and behavioral impairments, leading to tau truncation in NFTs. Tau P301S-transgenic mice with deletion of the gene encoding AEP show substantially reduced tau hyperphosphorylation, less synapse loss and rescue of impaired hippocampal synaptic function and cognitive deficits. Mice infected with adeno-associated virus encoding an uncleavable tau mutant showed attenuated pathological and behavioral defects compared to mice injected with adeno-associated virus encoding tau P301S. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.
KGaA, Weinheim Protein–ligand interactions serve as fundamental regulators of numerous biological processes. Among protein–ligand pairs, glycan binding proteins (GBPs) and the glycans they recognize represent unique and highly complex interactions implicated in a broad range of regulatory activities. With few exceptions, cell surface receptors and secreted proteins are heavily glycosylated. As these glycans often represent highly regulatable post-translational modifications, alterations in glycosylation can fundamentally impact GBP recognition. Among GBPs, galectins in particular appear to engage a diverse set of glycan determinants to impact a broad range of biological processes. In this review, we will explore factors that impact galectin activity, including the effect of glycan modification on galectin–glycan interactions.
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and neuropathological changes, including the deposition of amyloid β (Aβ) in senile plaques. The mechanisms causing the disease and Aβ accumulation are not well understood, but important genetic associations with apolipoprotein E genotype and involvement of lipoprotein receptors have become apparent. LR11 (also known as SorLA), a member of the low-density lipoprotein receptor family, has been identified previously as an altered transcript in microarray analyses of samples from human AD cases. Here, we show neuronal expression of the lipoprotein receptor LR11 in control brain in regions vulnerable to AD neuropathology and marked reduction of LR11 expression in these regions in AD brains before cell death. Overexpression of LR11 drastically reduces levels of extracellular Aβ and also lowers levels of total cellular amyloid precursor protein (APP). LR11 colocalizes with APP and regulates its trafficking in endocytic compartments, which are important intracellular sites for APP processing and Aβ generation. Endogenous LR11 localizes to neuronal multivesicular bodies in both rat and human brain. The robust correlation between reduced LR11 expression and AD neuropathology and its potent effects on extracellular Aβ levels suggest that this neuronal lipoprotein receptor could play an important role in AD pathogenesis.
Summary
Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.