Accumulation Of Proteins In Cytoplasm Research Articles

Alzheimer Type I Astrocytes: Still Mysterious Cells.

Over 100 years ago, von Hösslein and Alzheimer described enlarged and multinucleated astrocytes in the brains of patients with Wilson disease. These odd astrocytes, now well known to neuropathologists, are present in a large variety of neurological disorders, and yet the mechanisms underlying their generation and their functional attributes are still not well understood. They undergo abnormal mitoses and fail to accomplish cytokinesis, resulting in multinucleation. Oxidative stress, hypoxia, and inflammation may be contributing pathologies to generate these astrocytes. The abnormal mitoses occur from changes in cell shape, the accumulation of cytoplasmic proteins, and the mislocalization of many of the important molecules whose coordination is necessary for proper mitotic spindle formation. Modern technologies will be able to characterize their abnormalities and solve century old questions of their form and function.

Identification and functional analysis of two novel connexin 50 mutations associated with autosome dominant congenital cataracts.

Autosomal dominant congenital cataracts (ADCC) are clinically and genetically heterogeneous diseases. The present study recruited two Chinese families with bilateral nuclear cataract or zonular pulverulent phenotype. Direct sequencing of candidate genes identified two novel missense mutations of Cx50, Cx50P59A (c.175C > G) and Cx50R76H (c.227G > A), both co-segregated well with all affected individuals. Bioinformatics analysis predicted deleterious for both mutations. Functional and cellular behaviors of wild type and mutant Cx50 examined by stably transfecting recombinant systems revealed similar protein expression levels. Protein distribution pattern by fluorescence microscopy showed that Cx50R76H localized at appositional membranes forming gap junctions with enormous cytoplasmic protein accumulation, whereas the Cx50P59A mutation was found inefficient at forming detectable plaques. Cell growth test by MTT assay showed that induction of Cx50P59A decreased cell viability. Our study constitutes the first report that the Cx50P59A and Cx50R76H mutations are associated with ADCC and expands the mutation spectrum of Cx50 in association with congenital cataracts. The genetic, cellular, and functional data suggest that the altered intercellular communication governed by mutated Cx50 proteins may act as the molecular mechanism underlying ADCC, which further confirms the role of Cx50 in the maintenance of human lens transparency.

Restriction of protein synthesis abolishes senescence features at cellular and organismal levels.

Cellular senescence or its equivalence is induced by treatment of cells with an appropriate inducer of senescence in various cell types. Mild restriction of cytoplasmic protein synthesis prevented induction of all aspects of cellular senescence in normal and tumor-derived human cells. It allowed the cells to continuously grow with no sign of senescent features in the presence of various inducers. It also delayed replicative senescence in normal human fibroblasts. Moreover, it allowed for growth of the cells that had entered a senescent state. When adult worms of the nematode C. elegans were grown under protein-restricted conditions, their average and maximal lifespans were significantly extended. These results suggest that accumulation of cytoplasmic proteins due to imbalance in macromolecule synthesis is a fundamental cause of cellular senescence.

Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

Dominant mutations in FUS/TLS cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for aggregation of FUS in vitro and in vivo. Spontaneous in vitro formation of amyloid-like fibrillar aggregates was observed in mutant but not wild-type FUS, and notably, those fibrils functioned as efficient seeds to trigger the aggregation of wild-type protein. In addition, the G156E mutation did not disturb the nuclear localization of FUS but facilitated the formation of intranuclear inclusions in rat hippocampal neurons with significant cytotoxicity. We thus propose that intranuclear aggregation of FUS triggered by a subset of pathogenic mutations is an alternative pathomechanism of FUS-related fALS diseases.

A new perspective on estrogen receptor beta in ovarian cancer progression

O the gynecological malignancies, epithelial ovarian cancer is the leading cause of death in the great majority of developed countries with more than 140,000 women dying from this cancer across the world in 2008. The importance of estrogen signaling in the development and progression of ovarian cancer has been assumed to be less significant than in breast or endometrial malignancies, although preclinical studies and clinical data have shown that not only normal ovaries but also many malignant ovarian tumors can be considered as endocrine related, and hormone-dependent. Estrogens exert their action through two estrogen receptors (ERα and ERβ) that are encoded by separate genes. In the healthy ovary, the levels of ERβ are high and predominate over ERα, being ERβ1, -β2, and -β5 the most represented isoforms. The majority of ovarian tumors also express ERβ, but few studies have examined the prognostic role of ERβ isoforms in ovarian cancer. We have recently shown a strong statistical association of cytoplasmic ERβ2 immunoreactivity with an unfavorable outcome in advanced serous ovarian cancer patients, a finding also reported in familial and sporadic breast cancer. The functional role of cytoplasmic ERβ2 is far from being elucidated, however preliminary data from our ongoing studies suggest that cytoplasmic protein accumulation may confer pro-survival advantages to ovarian cancer cells by inhibiting apoptotic pathways.

A Unique Carboxyl-terminal Insert Domain in the Hematopoietic-specific, GTPase-deficient Rho GTPase RhoH Regulates Post-translational Processing

RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family that was first identified as a hypermutable gene in human B lineage lymphomas. RhoH remains in a constitutively active state and thus its effects are regulated by expression levels or post-translational modifications. Similar to other small GTPases, intracellular localization of RhoH is dependent upon the conserved "CAAX" box and surrounding sequences within the carboxyl (C) terminus. However, RhoH also contains a unique C-terminal "insert" domain of yet undetermined function. RhoH serves as adaptor molecule in T cell receptor signaling and RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia. Disease progression is attenuated in a Rhoh(-/-) mouse model of chronic lymphocytic leukemia and treatment of primary human chronic lymphocytic leukemia cells with Lenalidomide results in reduced RhoH protein levels. Thus, RhoH is a potential therapeutic target in B cell malignancies. In the current studies, we demonstrate that deletion of the insert domain (LFSINE) results in significant cytoplasmic protein accumulation. Using inhibitors of degradation pathways, we show that LFSINE regulates lysosomal RhoH uptake and degradation via chaperone-mediated autophagy. Whereas the C-terminal prenylation site is critical for ZAP70 interaction, subcellular localization and rescue of the Rhoh(-/-) T cell defect in vivo, the insert domain appears dispensable for these functions. Taken together, our findings suggest that the insert domain regulates protein stability and activity without otherwise affecting RhoH function.

P62/sequestosome 1 as a regulator of proteasome inhibitor‐induced autophagy in human retinal pigment epithelial cells

Abstract Purpose The pathogenesis of age‐related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin‐proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress –induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates.The functional roles of p62 and HSP70 were evaluated in conjunction with protesome inhibitor ‐induced autophagy in human RPE cells (ARPE‐19). Methods The p62, HSP70 and ubiquitin protein levels and localization were analyzed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results Proteasome inhibition evoked the accumulation of p62 and HSP70 that strongly co‐localized with each other in perinuclear aggregates. The p62 accumulation was time and concentration dependent after MG‐132 proteasome inhibitor loading. Interestingly, autophagy induction was p62 and Hsp70 independent. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently we showed that hsp70 mRNA depletion increased cell death in ARPE‐19 cells. Here we now demonstrate that p62 mRNA silencing has similar effects on cellular viability. Conclusion The p62 and HSP70 are central molecules in the regulation of protein turnover in human retinal pigment epithelial cells in proteasome inhibitor‐ induced autophagy.

Mutations in neutrophil elastase causing congenital neutropenia lead to cytoplasmic protein accumulation and induction of the unfolded protein response

Severe congenital neutropenia (SCN) and cyclic neutropenia (CyN) are sporadic or inherited hematologic disorders of myelopoiesis. Heterozygous mutations in the gene encoding neutrophil elastase (ELA2) have been reported in both diseases. We used an inducible system to express a panel of ELA2 mutations and found for almost all mutants disruption of intracellular neutrophil elastase (HNE) protein processing at different levels. This disruption resulted in cytoplasmic accumulation of a nonfunctional protein, thereby preventing its physiologic transport to azurophil granules. Furthermore, the secretory capacity of the mutant proteins was greatly diminished, indicating alteration of the regulated and the constitutive pathways. Through analysis of primary granulocytes from SCN patients carrying ELA2 mutations, we found an identical pattern of intracellular accumulation of mutant HNE protein in the cytoplasm. Moreover, cells expressing mutant HNE protein exhibited a significant increase in apoptosis associated with up-regulation of the master ER chaperone BiP, indicating that disturbance of intracellular trafficking results in activation of the mammalian unfolded protein response.

Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals

Peroxisomes are degraded by autophagic machinery termed "pexophagy" in yeast; however, whether this is essential for peroxisome degradation in mammals remains unknown. Here we have shown that Atg7, an essential gene for autophagy, plays a pivotal role in the degradation of excess peroxisomes in mammals. Following induction of peroxisomes by a 2-week treatment with phthalate esters in control and Atg7-deficient livers, peroxisomal degradation was monitored within 1 week after discontinuation of phthalate esters. Although most of the excess peroxisomes in the control liver were selectively degraded within 1 week, this rapid removal was exclusively impaired in the mutant liver. Furthermore, morphological analysis revealed that surplus peroxisomes, but not mutant hepatocytes, were surrounded by autophagosomes in the control. Our results indicated that the autophagic machinery is essential for the selective clearance of excess peroxisomes in mammals. This is the first direct evidence for the contribution of autophagic machinery in peroxisomal degradation in mammals.

Loopy Sec61 mutants

Sec61 forms the pore through which proteins enter the ER. On page 67, Cheng et al. show that its cytoplasmic face makes at least two contributions to translocation before the protein passage event.The predicted structure of yeast Sec61 suggests that several conserved charged residues, which are in cytoplasmic loops that link adjacent membrane-spanning domains, may interact with the ribosome during cotranslational translocation. The authors mutated many of these candidate residues within Sec61. Several of the mutations blocked cotranslational protein translocation in vivo, but the particular effect depended on which loop was mutated. Figure Mutations in L6 (blue) and L8 (white) define contact points (red) for cytoplasmic partners of Sec61. Point mutations in loop 8 (L8) interfered with the binding affinity between the ribosomal large subunit and the pore. Without this contact, normally ER-localized proteins accumulated in the cytoplasm. Cytoplasmic protein accumulation was also seen when loop 6 (L6) was mutated, although the binding affinity of the L6 mutants for the ribosome was not affected, suggesting that the cytoplasmic face of Sec61 has yet another function in translocation. This function might be to improve the efficiency of ribosome binding by signaling that a channel is unoccupied. Before transfering to the translocon, the ribosome first binds to the signal recognition particle (SRP) and its receptor (SR). The authors hypothesize that interactions between an unoccupied L6 and the SR might place the SR-SRP-ribosome intermediate near open channels and thus hasten its transfer. They are now looking for definitive evidence of L6–receptor interactions.

Hepatitis C virus core protein selectively inhibits synthesis and accumulation of p21/Waf1 and certain nuclear proteins.

By using a vaccinia virus-T7 expression system, possible effects of hepatitis C virus (HCV) core protein on synthesis and accumulation of host cellular proteins transiently expressed in cultured cells were analyzed. Immunoblot and immunofluorescence analyses revealed that synthesis and accumulation of certain nuclear proteins, such as p21/Waf1, p53, proliferating cell nuclear antigen and c-Fos, were strongly inhibited by HCV core protein. On the other hand, synthesis and accumulation of cytoplasmic proteins, such as 2'-5'-oligoadenylate synthetase (2'-5'-OAS), RNase L and MEK1, were barely affected by HCV core protein. Northern blot analysis showed that the degrees of mRNA expression for those proteins did not differ between HCV core protein-expressing cells and the control, suggesting that the inhibition occurred at the post-transcription level. Pulse-labeling analysis suggested that HCV core protein strongly inhibited synthesis of p21/Waf1 at the translation level. Once being accumulated in the nucleus, p21/Waf1 stability was not significantly affected by HCV core protein. Mutants of HCV core protein C-terminally deleted by 18 or 41 amino acids (aa), which were localized almost exclusively in the nucleus, lost their ability to inhibit synthesis/accumulation of p21/Waf1 whereas another mutant C-terminally deleted by 8 aa still maintained the same properties (subcellular localization and the inhibitory effect) as the full-length HCV core protein of 191 aa. Taken together, our present results suggest that expression of HCV core protein in the cytoplasm selectively inhibits synthesis of p21/Waf1 and some other nuclear proteins at the translation level.

Age-related decline in histone H1 fraction in human diploid fibroblast cultures

The age-related increase in cell volume and nuclear size of cultured human diploid fibroblasts reflected the accumulation of proteins in cytoplasm and nuclei of growth-retarded fibroblasts. Determination of the amount of nuclear proteins, which were fractionated into 0.15 M NaCl-soluble proteins, 0.4 N H 2SO 4-extractable proteins and residual acidic proteins, indicated that age-related increase in nuclear proteins was due mainly to the accumulation of residual acidic proteins. However, electrophoretic fractionation of histones from various passages of fibroblast cultures on acid urea polyacrylamide gel revealed that the relative amount of H1 fraction decreased with in vitro aging. This was further confirmed by mixing experiments examining the distribution of radioactivity of the histones from cell mixtures of young and senescent cultures labeled with [ 3H]lysine or [ 14C]lysine. A pulse label and chase experiment indicated that the observed decrease in the amount of histone H1 was mainly due to decrease in synthesis of histone H1 in senescent human fibroblast cultures.

Nuclear transplantation and the control of gene activity in animal development.

The transplantation of nuclei from differentiated or determined somatic cells to enucleated frogs’ eggs consistently leads to a complete and clearly recognizable change of gene activity. Within 1 to 2 h of nuclear transfer, somatic nuclei have come to resemble in structure and metabolism the zygote nuclei of fertilized eggs. The change in gene activity therefore takes place very soon after nuclear transfer and results from an effect of egg cytoplasm. The induced change in gene activity is associated with a selective accumulation of cytoplasmic proteins in transplanted nuclei. Examples are given of various ways in which nuclear transplantation and microinjection can be used to elucidate the intracellular movement of proteins and the effect of known protein fractions on gene activity.

Nuclear and cytoplasmic protein accumulation in phastimulated human lymphocytes during blastogenesis

Nuclear and cytoplasmic protein accumulation in PHA stimulated human lymphocytes, as measured by nuclear and cytoplasmic mass increase, was determined by microinterferometry. Both nuclear and cytoplasmic mass increased manyfold during blastogenesis, the increase of cytoplasmic mass being relatively greater than that of nuclear mass. A considerable part of nuclear mass accumulation occurred before the initiation of DNA synthesis. This is different to nuclear mass increase in tissue cultured cells in logarithmic phase of growth, in which nuclear mass increases parallel to DNA synthesis. This difference is discussed in view of what is known about nuclear changes following stimulation of lymphocytes.

Localization of homologous plasma proteins in the human placenta by fluorescent antibody

Normal and abnormal human placentas, as well as specimens of hydatidiform mole and choriocarcinoma, have been surveyed for homologous plasma albumin and globulin by the fluorescent antibody technique. Protein has been found broadly dispersed throughout the stromal ground substance of the villi, decidua, and cord, as well as in the intervillous spaces. Slight specific localization has been noted in the trophoblastic cytoplasm, but not within nuclei. Syncytial knots have shown somewhat higher levels of cytoplasmic protein accumulation. Albumin and gamma globulin exhibited similar distribution, and all placental material, including that from a case of erythroblastosis fetalis, presented similar topography. Syncytial masses in the choriocarcinoma revealed intracytoplasmic localization, while the mole showed a general diffusion through the vesicular stroma. The findings do not indicate synthesis of blood protein by the human placenta, but rather point to a transfer of maternal albumin and globulin across the syncytium, perhaps by pinocytosis and subsequent discharge into the villous stroma.