Sorting of MHC Class II Molecules into Exosomes through a Ubiquitin-Independent Pathway

Major histocompatibility complex class II (MHC-II) molecules accumulate in exocytic vesicles, called exosomes, which are secreted by antigen presenting cells. These vesicles are released following the fusion of multivesicular bodies (MVBs) with the plasma membrane. The molecular mechanisms regulating cargo selection remain to be fully characterized. As ubiquitination of the MHC-II β-chain cytoplasmic tail has recently been demonstrated in various cell types, we sought to determine if this post-translational modification is required for the incorporation of MHC-II molecules into exosomes. First, we stably transfected HeLa cells with a chimeric HLA-DR molecule in which the β-chain cytoplasmic tail is replaced by ubiquitin. Western blot analysis did not indicate preferential shedding of these chimeric molecules into exosomes. Next, we forced the ubiquitination of MHC-II in class II transactivator (CIITA)-expressing HeLa and HEK293 cells by transfecting the MARCH8 E3 ubiquitin ligase. Despite the almost complete downregulation of MHC-II from the plasma membrane, these molecules were not enriched in exosomes. Finally, site-directed mutagenesis of all cytoplasmic lysine residues on HLA-DR did not prevent inclusion into these vesicles. Taken together, these results demonstrate that ubiquitination of MHC-II is not a prerequisite for incorporation into exosomes.     

Dynamic Protein Pathway Activation Mapping of Adipose-Derived Stem Cell Differentiation Implicates Novel Regulators of Adipocyte Differentiation

Next to embryonic stem cell research, adult stem cell research is providing a promising alternative for enhanced tissue regeneration and transplantation. The key biochemical networks controlling the differentiation processes regulating stem cell biology remain largely disputed and or undefined, contributing to a lack of knowledge of the principle phosphoregulatory events propagating signal transduction. To effectively monitor these events relative to adipocyte differentiation, this study utilized a high throughput reverse phase protein microarray platform and characterized adult adipose-derived stem cell (ASC) differentiation through the monitoring of ∼100 phosphospecific endpoints with 33 distinct time points examined across 14 days. This kinetic-based analysis showed time ordered signal transduction ultimately implicating pathways correlated with adipogenic differentiation. To further validate the causal significance of these network activations, pharmacological targeting was implemented to include the chemical inhibitors MAPK inhibitor PD169316, rapamycin, and HNMPA-(AM)₃ yielding partial or complete disruption of adipocytic differentiation, as noted by a decrease or lack of lipid formation within the mature adipocytes. Based on this analysis, v-crk sarcoma virus CT10 oncogene homolog (CRKII) and c-abl oncogene 1, non-receptor tyrosine kinase (c-ABL) were implicated as novel key regulators of adipocyte differentiation, with v-akt murine thymoma viral oncogene (AKT), mammalian target of rapamycin (mTOR), and SMAD family member (SMAD) pathways being implicated as secondary regulators. This dynamic molecular profiling provides a novel insight into the signaling architecture of mesenchymal stem cell differentiation and may be useful in the development of therapeutic modulators for clinical applications; in addition to advancing the collective understanding of key cellular processes, ultimately contributing to more confident stem cell manipulation.Recent breakthroughs in adult stem cell research have increased industry focus on the potency and availability of these cells. Adult stem cells exist in many tissue types, but adult adipose-derived stem cells (ASCs)¹ provide a particularly abundant source obtained via through minimally invasive techniques (liposuction) (1, 2, 3). This plastic-adherent, multipotent cell population is also known as adipose-derived stromal cells among other names, with the International Fat Applied Technology Society reaching a consensus to refer to them as ASCs (4). Despite extensive research on the differentiation of stem cells, the underpinning molecular mechanisms remain an enigma. During adipogenesis, terminal differentiation from a mesenchymal stem cell to a mature adipocyte is characterized by the ability to store triglycerides that can be mobilized as fuel for other organs, but the cellular signaling actuating this transformation remains predominately undefined (5, 6). A few well-established factors inducing differentiation are: high concentrations of insulin (resulting in stimulation of the insulin-like growth factor 1 receptor), glucocorticoid agonists, peroxisome proliferator-activated receptor γ (PPAR_γ) agonist, and agents that elevate cAMP (7, 8).Primarily research into the process of adipogenesis has focused on gene expression profiling with proteomic technologies being used only more recently. Despite the benefits of gene expression profiling (9, 10), it shows little correlation to protein levels and provides no insight into the timing of protein activation. This simple fact highlights the need for a systems biology approach. More specifically, the monitoring of key post-translational modification events such as phosphorylation is necessary to characterize the signaling architecture regulating differentiation (11, 12). These reversible kinase-driven phosphorylation events alter protein confirmation, ultimately affecting enzymatic activity and protein–protein interactions leading to an array of cellular events from differentiation to gene expression, thus encompassing signal transduction. Characterization of this broad-scale signaling architecture is necessary to provide a more finite depiction of the complex signaling events directing a given cellular phenotype and aid in the understanding of the repercussions of alterations in regulation (13, 14). Presently, phosphoproteomic analysis of cellular differentiation processes such as with mass spectrometry has been performed in a very limited number of independent time points that span the cellular differentiation process, and important dynamic changes in the cellular signaling architecture may have been missed (12, 15, 16). This study however, utilized the reverse phase protein microarray (RPMA) to maximize both the number of time points and phosphoproteins able to be examined simultaneously. RPMAs enable the quantitative interrogation of the phosphorylation state of hundreds of signaling proteins simultaneously for hundreds of cell lysates allowing for broad-scale pathway activation mapping analysis (17). This ultrasensitive platform has demonstrated sensitivity of as little as 1000 molecules per spot with less than 1/10th of a cell equivalent volume analyzed per spot and intraslide and interslide CV between 3–10% (18, 19). RPMAs greatly reduce the required sample size with spot deposits averaging between 0.3–2 nL, and yet show concurrent findings when parallel processing of samples via Western blot is performed (20, 21, 22, 23, 24).Despite the implication of various markers of adipogenesis, there remains little consensus on the overarching signal transduction governing the differentiation process. The objective of this research was to better characterize this process through utilization of ASCs combined with broad-scale protein pathway activation mapping, using a dynamic experimental design. Lineage signal transduction profiles were established through the monitoring of protein network activation during the course of differentiation into adipocyte, osteoblast, and chondrocyte lineages. This multilineage kinetic experimentation allowed for global examination of signal transduction through the monitoring of ∼100 phosphospecific endpoints, across 33 consecutive time points that spanned a 14 day period to reach terminal differentiation demonstrating time-specific and lineage-specific signaling. This experimental design allowed for isolation of a subset of time-specific endpoints unique to adipogenesis relative to the other lineages that could then be further tested for causal significance using pharmacologic knock-out analysis.     

A RecA Protein Surface Required for Activation of DNA Polymerase V

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3''-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3''-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA''₂B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.     

Chromosomal Localization of Human RNA Polymerase II Subunit Genes

The eukaryotic DNA-dependent RNA polymerase II (or B) is composed of 10 to 14 polypeptides ranging from 220 to 10 kDa. To gain further insight into the molecular structure and function of these subunits, we have undertaken the molecular cloning of nucleotide sequences corresponding to the human enzyme. The cDNAs of five subunits (hRPB220, hRPB140, hRPB33, hRPB25, and hRPB14.5) have been isolated. Using in situ hybridization, we show that the genes of these subunits have distinct chromosomal locations (17p13, 4q12, 16q13-q21, 19p13.3, and 19q12, respectively). Thus, if assembly of active polymerase molecules requires coordinated expression from these independent genes, mechanisms that ensure tight coregulation of the corresponding promoters must exist.     

Disappearance of a Novel Protein Component of the 26S Proteasome duringXenopusOocyte Maturation

We have prepared polyclonal antibodies againstXenopus20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development ofXenopus laevis.A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.