Intercellular exchange of proteins: The immune cell habit of sharing

The recent recognition of new types of cell-cell communication pathways challenges classic theories of cell autonomy. Evidence of functional “proteome mixing” among interacting cells, particularly immune cells, supports the notion that no cell is an island, and that even these “unsplittable” units are actually non-autonomous. We summarize various mechanisms of intercellular transfer of proteins—trans-endocytosis, trogocytosis, exosomal transport, shuttle through nanotubes, and cell-contact-dependent intercellular transfer of intracellular proteins including oncogenic Ras. These phenomena suggest exciting new possibilities for proteome research, focusing on system-level proteomics that characterize cell contents and functions in the context of intercellular protein transfer.     

Nonconventional trafficking of Ras associated with Ras signal organization

Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with 'rasosomes', randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function.     

Spatiotemporal Organization of Ras Signaling: Rasosomes and the Galectin Switch

Summary 1. Ras signaling and oncogenesis depend on the dynamic interplay of Ras with distinctive plasma membrane (PM) microdomains and various intracellular compartments. Such interaction is dictated by individual elements in the carboxy-terminal domain of the Ras proteins, including a farnesyl isoprenoid group, sequences in the hypervariable region (hvr)-linker, and palmitoyl groups in H/N-Ras isoforms.2. The farnesyl group acts as a specific recognition unit that interacts with prenyl-binding pockets in galectin-1 (Gal-1), galectin-3 (Gal-3), and cGMP phosphodiesterase δ. This interaction appears to contribute to the prolongation of Ras signals in the PM, the determination of Ras effector usage, and perhaps also the transport of cytoplasmic Ras. Gal-1 promotes H-Ras signaling to Raf at the expense of phosphoinositide 3-kinase (PI3-K) and Ral guanine nucleotide exchange factor (RalGEF), while galectin-3 promotes K-Ras signaling to both Raf and PI3-K.3. The hvr-linker and the palmitates of H-Ras and N-Ras determine the micro- and macro-localizations of these proteins in the PM and in the Golgi, as well as in ‘rasosomes’, randomly moving nanoparticles that carry palmitoylated Ras proteins and their signal through the cytoplasm.4. The dynamic compartmentalization of Ras proteins contributes to the spatial organization of Ras signaling, promotes redistribution of Ras, and provides an additional level of selectivity to the signal output of this regulatory GTPase.     

Inhibition of matrilysin expression by antisense or RNA interference decreases lysophosphatidic acid-induced epithelial ovarian cancer invasion

Our previous reports show that matrilysin [matrix metalloproteinase (MMP)-7] is overexpressed in epithelial ovarian cancer (EOC) and recombinant MMP-7 promotes EOC invasion in vitro. In the present study, we further evaluated the correlation of MMP-7 expression to EOC invasiveness and examined its role in lysophosphatidic acid (LPA)-induced invasion. By sense and antisense gene transfection in vitro, we show that overexpression of MMP-7 in all MMP-7 stably transfected DOV13 clones significantly enhanced their invasiveness, although MMP-7 antisense transfection caused a 91% decrease of MMP-7 expression (P < 0.01) and 87% decrease of invasion (P < 0.05) in geneticin (G418)-selected DOV13 clone P47-M7As-3 compared with vector-transfected control. As assessed by MMP-7 ELISA, LPA treatment at 10 to 80 micromol/L significantly stimulated the secretion of total MMP-7 in DOV13 conditioned medium (P < 0.01). In addition, LPA apparently induced the activation of MMP-7 in DOV13 cells as detected by gelatin zymography. In the antisense MMP-7-transfected DOV13 clone (P47-M7As-3), LPA-increased invasion was significantly decreased compared with vector control. Moreover, knocking down of MMP-7 by small interfering RNA also suppressed LPA-induced invasion in two EOC cell lines (DOV13 and R182). Altogether, our results show that MMP-7 expression is correlated with EOC invasiveness and LPA-induced MMP-7 secretion/activation may represent a new mechanism that facilitates ovarian cancer invasion besides the well-known induction of MT1-MMP-mediated proMMP-2 activation by LPA.     

Double freezing of bovine semen

Fertility of bull spermatozoa cryopreserved in large volume by directional freezing technique, thawed, repackaged in straws and refrozen over liquid nitrogen vapor (double freezing, DF) was compared to conventional single freezing in straws (CF). Semen was collected from 6 bulls, 4 of which were selected for the field trial. Each semen collection was split into two parts, one frozen by CF and the other by DF. In vitro semen evaluations included motility (fresh, upon thawing and after 3 h incubation at 37 °C), viability and acrosome integrity. A total of 3610 cows and heifers were randomly inseminated by either CF or DF at about equal numbers. In vitro sperm analysis indicated no difference between CF and directional freezing in large volume and both were superior to DF (P < 0.001). Between-bull variations in fresh semen and in their reaction to CF or DF were apparent. Logistic regression analysis revealed that freezing method, bull, parity and inseminating technician, all had significant effect on pregnancy outcome (P ≤ 0.001 for all). Conception rate (CR) was 32.98% for CF and 28.05% for DF. Only in one bull conception rate by CF was significantly superior to DF (P < 0.05). When divided into heifers, primi- and pluriparous cows, only the difference in CR between the pluriparous cows was significant (P = 0.005). In conclusion, acceptable CR can be achieved by DF technique. These can be improved by selecting suitable bulls. The DF technique can be utilized in storage, sperm sexing and genome resource banking.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.