A review of the effects of the cell environment physicochemical nanoarchitecture on stem cell commitment

Physicochemical features of a cell nanoenvironment exert important influence on stem cell behavior and include the influence of matrix elasticity and topography on differentiation processes. The presence of growth factors such as TGF-β and BMPs on these matrices provides chemical cues and thus plays vital role in directing eventual stem cell fate. Engineering of functional biomimetic scaffolds that present programmed spatio-temporal physical and chemical signals to stem cells holds great promise in stem cell therapy. Progress in this field requires tacit understanding of the mechanistic aspects of cell-environment nanointeractions, so that they can be manipulated and exploited for the design of sophisticated next generation biomaterials. In this review, we report and discuss the evolution of these processes and pathways in the context of matrix adhesion as they might relate to stemness and stem cell differentiation. Super-resolution microscopy and single-molecule methods for in vitro nano-manipulation are helping to identify and characterize the molecules and mechanics of structural transitions within stem cells and matrices. All these advances facilitate research toward understanding of stem cell niche and consequently to developing new class of biomaterials helping the “used biomaterials” for applications in tissue engineering and regenerative medicine.     

Ubiquitin not only serves as a tag but also assists degradation by inducing protein unfolding

Protein ubiquitination controls the cellular fate of numerous eukaryotic proteins. Despite its importance, many fundamental questions remain regarding its mechanism. One such question is how ubiquitination alters the biophysical properties of the modified protein and whether these alterations are significant in the cellular context. In this study, we investigate the effects of ubiquitination on the folding thermodynamics and mechanism of various substrates using computational tools and find that ubiquitination changes the thermal stability of modified proteins in a manner relevant to cellular processes. These changes depend on the substrate modification site and on the type of ubiquitination. Ubiquitination of the substrate Ubc7 at the residues that are modified in vivo prior to proteasomal degradation uniquely results in significant thermal destabilization and a local unwinding near the modification site, which indicates that ubiquitination possibly facilitates the unfolding process and improves substrate degradation efficiency. With respect to the substrate p19^4inkd, our results support a synergetic effect of ubiquitination and phosphorylation on the degradation process via enhanced thermal destabilization. Our study implies that, in addition to its known role as a recognition signal, the ubiquitin attachment may be directly involved in the cellular process it regulates by changing the biophysical properties of the substrate.     

Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC₅₀ to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.     

Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F(0), and proteolytically cleaved into the mature F(1) and F(2) heterodimer, following an HDLVDGVK(109) motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK(109) motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK(109) motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein.     

Immature properties of large-conductance calcium-activated potassium channels in rat neuroepithelium

The pharmacological and biophysical properties of large-conductance Ca-activated K (BK) channels from embryonic rat telencephalic neuroepithelium were investigated with in situ patch-clamp techniques. A fraction of these channels exhibited properties characteristic of BK channels recorded in well differentiated cells, including normal gating mode (BK_N channels). The vast majority of BK channels expressed distinctive properties, the most conspicuous being their buzz gating mode (BK_B channels). BK_B channels were insensitive to a concentration of charybdotoxin that completely and reversibly blocked BK_N channels. In contrast with the strict dependence of BK_N channel activation on cytoplasmic Ca, BK_B channels displayed substantially high open probability (P _o) after inside-out patch excision in a Ca-free medium. Intracellular trypsin down-regulated the P _o of BK_B channels, which then exhibited a greater sensitivity to cytoplasmic Ca, mainly in the positive direction (increased P _o with increased Ca). This suggested a modulatory role for Ca as opposed to its gating role in BK_N channels. Ca ions also reduced current amplitude of both types of channels. BK_B channels were less voltage sensitive than BK_N channels, but this was not correlated with their lower Ca sensitivity. We speculate that BK_B channels may represent immature forms in the developmental expression of BK channels. Received: 1 August 1995 /Received after revision: 24 October 1995 /Accepted: 25 October 1995     

Post-translational modifications: Regulators of neurodegenerative proteinopathies

One of the hallmark features in the neurodegenerative disorders (NDDs) is the accumulation of aggregated and/or non-functional protein in the cellular milieu. Post-translational modifications (PTMs) are an essential regulator of non-functional protein aggregation in the pathogenesis of NDDs. Any alteration in the post-translational mechanism and the protein quality control system, for instance, molecular chaperone, ubiquitin-proteasome system, autophagy-lysosomal degradation pathway, enhances the accumulation of misfolded protein, which causes neuronal dysfunction. Post-translational modification plays many roles in protein turnover rate, accumulation of aggregate and can also help in the degradation of disease-causing toxic metabolites. PTMs such as acetylation, glycosylation, phosphorylation, ubiquitination, palmitoylation, SUMOylation, nitration, oxidation, and many others regulate protein homeostasis, which includes protein structure, functions and aggregation propensity. Different studies demonstrated the involvement of PTMs in the regulation of signaling cascades such as PI3K/Akt/GSK3β, MAPK cascade, AMPK pathway, and Wnt signaling pathway in the pathogenesis of NDDs. Further, mounting evidence suggests that targeting different PTMs with small chemical molecules, which acts as an inhibitor or activator, reverse misfolded protein accumulation and thus enhances the neuroprotection. Herein, we briefly discuss the protein aggregation and various domain structures of different proteins involved in the NDDs, indicating critical amino acid residues where PTMs occur. We also describe the implementation and involvement of various PTMs on signaling cascade and cellular processes in NDDs. Lastly, we implement our current understanding of the therapeutic importance of PTMs in neurodegeneration, along with emerging techniques targeting various PTMs.     

辛二酰苯胺异羟肟酸对人肝星状细胞增殖和凋亡的影响

目的研究组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸(SAHA)对人肝星状细胞系LX-2增殖及凋亡的影响及其可能机制。方法体外应用SAHA作用于LX-2细胞,倒置显微镜观察LX-2细胞形态,MTT法检测细胞增殖;荧光显微镜及流式细胞仪Annexin V-FITC/PI法检测细胞凋亡率;Western blot检测α-SMA、Ⅰ型胶原、ac H3K9、ac H3K14和ac H3K18蛋白表达。结果 SAHA呈剂量依赖性显著抑制LX-2细胞增殖(P0.05);SAHA对LX-2细胞凋亡具有呈时间依赖性的促进作用(P0.05);SAHA处理LX-2细胞后,α-SMA和Ⅰ型胶原蛋白表达水平明显降低(P0.05),而ac H3K9、ac H3K14和ac H3K18乙酰化修饰水平明显升高(P0.01)。结论 SAHA抗肝纤维化的机制可能与下调α-SMA及Ⅰ型胶原蛋白表达,上调组蛋白ac H3K9、ac H3K14和ac H3K18乙酰化修饰水平有关。     

Partial Blocking of Mouse DSPP Processing by Substitution of Gly451–Asp452 Bond Suggests the Presence of Secondary Cleavage Site(s)

Dentin sialophosphoprotein (DSPP) in the extracellular matrix of dentin is cleaved into dentin sialoprotein and dentin phosphoprotein, which originate from the NH₂-terminal and COOH-terminal regions of DSPP, respectively. In the proteolytic processing of mouse DSPP, the peptide bond at Gly⁴⁵¹–Asp⁴⁵² has been shown to be cleaved by bone morphogenetic protein 1 (BMP1)/Tolloid-like metalloproteinases. In this study, we generated transgenic mice expressing a mutant DSPP in which Asp⁴⁵² was substituted by Ala⁴⁵². Protein chemistry analyses of extracts from the long bone of these transgenic mice showed that the D452A substitution partially blocked DSPP processing in vivo. When the full-length form of mutant DSPP (designated “D452A-DSPP”) isolated from the transgenic mice was treated with BMP1 in vitro, a portion of the D452A-DSPP was cleaved, suggesting the presence of secondary peptide bond(s) that can be broken by BMP1. To identify the potential secondary DSPP cleavage site(s), site-directed mutagenesis was performed to generate nine DNA constructs expressing DSPP-bearing substitutions at potential scission sites. These different types of mutant DSPP made in eukaryotic cell lines were treated with BMP1 and the digestion products were assessed by Western immunoblotting. All of the mutant DSPP molecular species were partially cleaved by BMP1, giving rise to a protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to that of normal dentin sialoprotein. Taken together, we concluded that in addition to the peptide bond Gly⁴⁵¹–Asp⁴⁵², there must be a cryptic cleavage site or sites close to Asp⁴⁵² in the mouse DSPP that can be cleaved by BMP1.