Modulation of autophagy as new approach in mesenchymal stem cell-based therapy.

Due to their trophic and immunoregulatory characteristics mesenchymal stem cells (MSCs) have tremendous potential for use in a variety of clinical applications. Challenges in MSCs' clinical applications include low survival of transplanted cells and low grafting efficiency requiring use of a high number of MSCs to achieve therapeutic benefits. Accordingly, new approaches are urgently needed in order to overcome these limitations. Recent evidence indicates that modulation of autophagy in MSCs prior to their transplantation enhances survival and viability of engrafted MSCs and promotes their pro-angiogenic and immunomodulatory characteristics. Here, we review the current literature describing mechanisms by which modulation of autophagy strengthens pro-angiogenic and immunosuppressive characteristics of MSCs in animal models of multiple sclerosis, osteoporosis, diabetic limb ischemia, myocardial infarction, acute graft-versus-host disease, kidney and liver diseases. Obtained results suggest that modulation of autophagy in MSCs may represent a new therapeutic approach that could enhance efficacy of MSCs in the treatment of ischemic and autoimmune diseases.

Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes.

Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains.

Despite having high-resolution structures for eukaryotic large ribosomal subunits, it remained unclear how these ribonucleoprotein complexes are constructed in living cells. Nevertheless, knowing where ribosomal proteins interact with ribosomal RNA (rRNA) provides a strategic platform to investigate the connection between spatial and temporal aspects of 60S subunit biogenesis. We previously found that the function of individual yeast large subunit ribosomal proteins (RPLs) in precursor rRNA (pre-rRNA) processing correlates with their location in the structure of mature 60S subunits. This observation suggested that there is an order by which 60S subunits are formed. To test this model, we used proteomic approaches to assay changes in the levels of ribosomal proteins and assembly factors in preribosomes when RPLs functioning in early, middle, and late steps of pre-60S assembly are depleted. Our results demonstrate that structural domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical fashion. Assembly begins at the convex solvent side, followed by the polypeptide exit tunnel, the intersubunit side, and finally the central protuberance. This model provides an initial paradigm for the sequential assembly of eukaryotic 60S subunits. Our results reveal striking differences and similarities between assembly of bacterial and eukaryotic large ribosomal subunits, providing insights into how these RNA-protein particles evolved.

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing.

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits.

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA(3) to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA(3) pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A(3) assembly factors and for proper assembly of the neighborhoods containing domains I and II.

Kinesin molecular motor Eg5 functions during polypeptide synthesis.

The kinesin-related molecular motor Eg5 plays roles in cell division, promoting spindle assembly. We show that during interphase Eg5 is associated with ribosomes and is required for optimal nascent polypeptide synthesis. When Eg5 was inhibited, ribosomes no longer bound to microtubules in vitro, ribosome transit rates slowed, and polysomes accumulated in intact cells, suggesting defects in elongation or termination during polypeptide synthesis. These results demonstrate that the molecular motor Eg5 associates with ribosomes and enhances the efficiency of translation.

Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes.

More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors--e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs--remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm.

Effect of dietary intake of phytoestrogens on estrogen receptor status in premenopausal women with breast cancer.

Although many dietary studies have focused on breast cancer risk, few have examined dietary influence on tumor characteristics such as estrogen receptor (ER) status. Because phytoestrogens may modulate hormone levels and ER expression, we analyzed ER status and phytoestrogen intake in a case-case study of 124 premenopausal breast cancer patients. We assessed intake with a food-frequency questionnaire and obtained ER status from medical records. Rather than focusing on risk, we evaluated whether low intakes were more strongly associated with ER-negative tumors than with ER-positive disease. In logistic regression adjusting for potential confounders, threefold greater risks of ER-negative tumors relative to ER-positive tumors were associated with low intake of the isoflavones genistein (odds ratio, OR=3.50; 95% confidence interval, CI=1.43-8.58) and daidzein (OR=3.10; 95% CI=1.31-7.30). Low intake of the flavonoid kaempferol (OR=0.36; 95% CI=0.16-0.83), the trace element boron (OR=0.33; 95% CI=0.13-0.83), and the phytosterol beta-sitosterol (OR=0.42; 95% CI=0.18-0.98) were associated with decreased risk of ER-negative tumors relative to ER-positive disease. Other phytoestrogens were not significantly associated with ER status. Thus, in premenopausal patients, some phytoestrogens may affect breast carcinogenesis by influencing ER status. Such findings suggest new directions for mechanistic research on dietary factors in breast carcinogenesis that may have relevance for prevention and clinical treatment.

Dietary intake of selected fatty acids, cholesterol and carotenoids and estrogen receptor status in premenopausal breast cancer patients.

Although a wealth of research has focused on the influence of diet on breast cancer risk, the relationships between dietary factors and tumor characteristics of breast cancer, like estrogen receptor (ER) status, are not well characterized. In a case-case study, we evaluated self-reported dietary intake for five individual carotenoids, selected fatty acids, and cholesterol 1 year before diagnosis in 34 premenopausal breast cancer patients with ER-negative tumors and 86 premenopausal breast cancer patients with ER-positive tumors from The University of Texas M. D. Anderson Cancer Center. In multivariate logistic regression analysis adjusted for age, body mass index, and ethnicity, high intakes of linoleic acid were associated with more than a threefold greater risk of ER-negative disease than ER-positive disease (odds ratio (OR) = 3.48, 95% confidence interval (CI) = 1.42-8.54), whereas high cholesterol intake was associated with lower risk of ER-negative disease (OR = 0.35, 95% CI = 0.14-0.92). In a model evaluating carotenoids, selected fatty acids, and cholesterol together, the association with high intake of linoleic acid remained statistically significant (OR = 3.96,95% CI = 1.53-10.25), while those for high intake of cholesterol (OR = 0.38, 95% CI = 0.14-1.03) and low intake of cryptoxanthin (OR = 0.43, 95% CI = 0.17-1.06) were of marginal significance. While no striking associations were observed for the intakes of total carotenoids, selected fatty acids, and cholesterol, our analysis revealed an association for the consumption of a specific fatty acid (i.e., linoleic acid), suggesting dietary influence of this factor on ER status in premenopausal breast cancer patients. However, larger studies are needed to clarify the role of micronutrients in ER status in breast cancer.

Saccharomyces cerevisiae nucleolar protein Nop7p is necessary for biogenesis of 60S ribosomal subunits.

To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A.