Site-specific protein modification on living cells catalyzed by Sortase

The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incubation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering.     

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.     

The PASTA server for protein aggregation prediction

Many different proteins aggregate into amyloid fibrils characterized by cross-beta structure. beta-strands contributed by distinct protein molecules are generally found in a parallel in-register alignment. Here, we describe the web server for a novel algorithm, prediction of amyloid structure aggregation (PASTA), to predict the most aggregation-prone portions and the corresponding beta-strand inter-molecular pairing for a given input sequence. PASTA was previously shown to yield results in excellent agreement with available experimental observations, when tested on both natively unfolded and structured proteins. The web server and downloadable source code are freely accessible from the URL: http://protein.cribi.unipd.it/pasta/.     

A need for speed: genetic encoding of rapid cycloaddition chemistries for protein labelling in living cells

Rapid reactions: Several reactants for strain-promoted cycloaddition reactions have been genetically encoded as the side chains of noncanonical amino acids. This results in decisive improvements for the fluorescent labelling of intracellular proteins such as quantitative turnover, completion of labelling reactions within minutes, fluorogenic effects and even partial orthogonality for multicolour labelling.     

Effectiveness factor for spatial gradient sensing in living cells

We consider the steady-state pattern of messenger molecules produced in the membrane of a cell perceiving and responding to an extracellular gradient of chemoattractant, which directs cell movement towards the chemoattractant source. Specifically, we analyze the undesirable effect of lateral diffusion in blurring the intracellular messenger profile. The concept of an effectiveness factor, akin to the analysis of reactions in porous catalysts, is applied to the spatial gradient sensing problem, with the distinction that slow, not fast, diffusion is required for effective gradient sensing. Analytical effectiveness factor expressions are derived for ideal geometries and then generalized to arbitrary cell shapes. In the case of mouse fibroblasts responding to gradients of platelet-derived growth factor, we conclude that the cell morphology and orientation with respect to the gradient can dictate whether messenger diffusion obliterates gradient sensing or has very little effect. The analysis outlined here allows the effect of intracellular messenger diffusion on spatial gradient sensing to be quantified for individual cells.     

Role of prion protein aggregation in neurotoxicity

In several neurodegenerative diseases, such as Parkinson, Alzheimer's, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.     

Rhodamine-based fluorescent sensor for mercury in buffer solution and living cells

A novel fluorescent sensor based on thiooxorhodamine B has been prepared to detect Hg²⁺ in aqueous buffer solution. It demonstrates high selectivity for sensing Hg²⁺ with about 383-fold enhancement in fluorescence emission intensity and micromolar sensitivity (K_d = 7.5 × 10⁻⁶ mol L⁻¹) in comparison with alkali and alkaline earth metal ions (K⁺, Na⁺, Mg²⁺, Ca²⁺) and other transition metal ions (Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ag⁺, Pb²⁺, Cr³⁺, Fe³⁺). Meanwhile the distinct color changes and rapid switch-on fluorescence also provide ‘naked eyes’ detection for Hg²⁺ over a broad pH range. Moreover, such sensor is cell-permeable and can visualize the changes of intracellular mercury ions in living cells using fluorescence microscopy.     

Role of water in protein aggregation and amyloid polymorphism

A variety of neurodegenerative diseases are associated with amyloid plaques, which begin as soluble protein oligomers but develop into amyloid fibrils. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Mechanisms of in vivo amyloid formation involve a number of coconspirators and complex interactions with membranes. Nevertheless, understanding the biophysical basis of simpler in vitro amyloid formation is considered important for discovering ligands that preferentially bind regions harboring amyloidogenic tendencies. The determination of the fibril structure of many peptides has set the stage for probing the dynamics of oligomer formation and amyloid growth through computer simulations. Most experimental and simulation studies, however, have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked in oligomer formation and assembly to protofilaments and amyloid fibrils. In this Account, we provide a perspective on how interactions with water affect folding landscapes of amyloid beta (Aβ) monomers, oligomer formation in the Aβ16-22 fragment, and protofilament formation in a peptide from yeast prion Sup35. Explicit molecular dynamics simulations illustrate how water controls the self-assembly of higher order structures, providing a structural basis for understanding the kinetics of oligomer and fibril growth. Simulations show that monomers of Aβ peptides sample a number of compact conformations. The formation of aggregation-prone structures (N*) with a salt bridge, strikingly similar to the structure in the fibril, requires overcoming a high desolvation barrier. In general, sequences for which N* structures are not significantly populated are unlikely to aggregate. Oligomers and fibrils generally form in two steps. First, water is expelled from the region between peptides rich in hydrophobic residues (for example, Aβ16-22), resulting in disordered oligomers. Then the peptides align along a preferred axis to form ordered structures with anti-parallel β-strand arrangement. The rate-limiting step in the ordered assembly is the rearrangement of the peptides within a confining volume. The mechanism of protofilament formation in a polar peptide fragment from the yeast prion, in which the two sheets are packed against each other and create a dry interface, illustrates that water dramatically slows self-assembly. As the sheets approach each other, two perfectly ordered one-dimensional water wires form. They are stabilized by hydrogen bonds to the amide groups of the polar side chains, resulting in the formation of long-lived metastable structures. Release of trapped water from the pore creates a helically twisted protofilament with a dry interface. Similarly, the driving force for addition of a solvated monomer to a preformed fibril is water release; the entropy gain and favorable interpeptide hydrogen bond formation compensate for entropy loss in the peptides. We conclude by offering evidence that a two-step model, similar to that postulated for protein crystallization, must also hold for higher order amyloid structure formation starting from N*. Distinct water-laden polymorphic structures result from multiple N* structures. Water plays multifarious roles in all of these protein aggregations. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow fibril growth rates in hydrophilic sequences.     

Rhodamine-based fluorescent sensor for mercury in buffer solution and living cells

A novel fluorescent sensor based on thiooxorhodamine B has been prepared to detect Hg²⁺ in aqueous buffer solution. It demonstrates high selectivity for sensing Hg²⁺ with about 383-fold enhancement in fluorescence emission intensity and micromolar sensitivity (K_d = 7.5 × 10⁻⁶ mol L⁻¹) in comparison with alkali and alkaline earth metal ions (K⁺, Na⁺, Mg²⁺, Ca²⁺) and other transition metal ions (Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ag⁺, Pb²⁺, Cr³⁺, Fe³⁺). Meanwhile the distinct color changes and rapid switch-on fluorescence also provide ‘naked eyes’ detection for Hg²⁺ over a broad pH range. Moreover, such sensor is cell-permeable and can visualize the changes of intracellular mercury ions in living cells using fluorescence microscopy.     

Chemical tags for labeling proteins inside living cells

To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, we now face the challenge of understanding how macromolecular machines, signaling pathways, and other biological networks operate in the complex environment of the living cell. The fluorescent proteins (FPs) revolutionized our ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biology, they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chemical methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chemical tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chemical tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an organic fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chemical tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, we focus on the development and application of chemical tags for studying protein function within living cells. Thus, in our overview of different chemical tagging strategies and technologies, we emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. We highlight recent applications in which the chemical tags have enabled sophisticated biophysical measurements that would be difficult or even impossible with FPs. Finally, we conclude by looking forward to (i) the development of high-photon-output chemical tags compatible with living cells to enable high-resolution imaging, (ii) the realization of the potential of the chemical tags to significantly reduce tag size, and (iii) the exploitation of the modular chemical tag label to go beyond fluorescent imaging.     

Peptide and protein mimetics inhibiting amyloid beta-peptide aggregation

Protein misfolding is related to some fatal diseases including Alzheimer's disease (AD). Amyloid beta-peptide (Abeta) generated from amyloid precursor protein can aggregate into amyloid fibrils, which are known to be a major component of Abeta deposits (senile plaques). The fibril formation of Abeta is typical of a nucleation-dependent process through self-recognition. Moreover, during fibrillization, several metastable intermediates such as soluble oligomers, including Abeta-derived diffusible ligands (ADDLs) and Abeta*56, are produced, which are thought to be the most toxic species to neuronal cells. Therefore, construction of molecules that decrease the Abeta aggregates, including soluble oligomers, protofibrils, and amyloid fibrils, might further our understanding of the mechanism(s) behind fibril formation and enable targeted drug discovery against AD. To this aim, various peptides and peptide derivatives have been constructed using the "Abeta binding element" based on the structural models of Abeta amyloid fibrils and the mechanisms of self-assembly. The central hydrophobic amino acid sequence, LVFF, of Abeta is a key sequence to self-assemble into amyloid fibrils. By combination of this core sequence with a hydrophobic or hydrophilic moiety, such as cholic acid or aminoethoxy ethoxy acetic acid units, respectively, good inhibitors of Abeta aggregation can be designed and synthesized. A peptide, LF, consisting of the sequence Ac-KQKLLLFLEE-NH 2, was designed based on the core sequence of Abeta but with a simplified amino acid sequence. The LF peptide can form amyloid-like fibrils that efficiently coassemble with mature Abeta1-42 fibrils. The LF peptide was also observed to immediately transform the soluble oligomers of Abeta1-42, which are thought to pose toxicity in AD, into amyloid-like fibrils. On the other hand, two Abeta-like beta-strands with a parallel orientation were embedded in green fluorescent protein (GFP), comprised of a beta-barrel structure, to make pseudo-Abeta beta-sheets on its surface. The GFP variant P13H binds to Abeta1-42 and inhibits Abeta1-42 oligomerization effectively in a substoichiometric condition. Thus, molecules capable of binding to Abeta can be designed based on structural similarities with the Abeta molecule. The peptide and protein mimetics based on the structural features of Abeta might lead to the development of drug candidates against AD.     

LTD蛋白体外对HeLa细胞毒性的影响

目的探讨LTD蛋白在体外对HeLa细胞毒性的影响。方法将不同浓度的LTD蛋白作用于HeLa细胞,光学显微镜下观察细胞形态等变化,MTT法检测细胞增殖情况,激光共聚焦显微镜观察蛋白在细胞内的定位,HE染色和TUNNEL试剂盒检测细胞凋亡情况。结果 LTD蛋白对HeLa细胞具有毒性作用,能抑制细胞增殖,引起细胞凋亡。随着蛋白浓度的增加及作用时间的延长,HeLa细胞凋亡现象明显,生长抑制作用增强,蛋白浓度与细胞A_(490)值之间明显相关;且LTD由定位于细胞膜变为定位于胞浆和细胞核。结论 LTD蛋白促进HeLa细胞凋亡,对其增殖具有明显的抑制作用。     

A highly sensitive turn-off fluorescent probe based on 2D Eu(III)-MOFs nanosheets for glutathione in vitro and living cells

Glutathione (GSH) is generally used as an effective and sensitive tumor marker because its abnormal levels are associated with high free radical level in tumor. In this work, GSH could be easily detected by a kind of designed Eu-based metal–organic frameworks (Eu-MOFs, named Eu(DTBA)) fluorescent sensor. Due to the “antenna effect” of 4,4′-dithiobenzoic acid (4,4′-DTBA) ligands on Eu³⁺, Eu(DTBA) emits the strong characteristic red light of Eu³⁺ under ultraviolet excitation. Moreover, the emission intensity strongly depends on GSH concentrations. Thus, Eu(DTBA) can serve as a turn-off fluorescent switch of GSH because its framework structure can easily be destroyed by GSH that leads to the fluorescence quenching. Remarkably, during sensing GSH, Eu(DTBA) has shown many appealing performances, such as broad a response window (0–20 mM), fast response (3 min), high sensitivity (LOD = 0.35 µM), and excellent anti-interference ability. The real bioimaging application has demonstrated that the reported Eu(DTBA) can be used as an excellent bioimaging agent to successfully distinguish tumor cells from normal cells in clinical diagnosis.     

Vertical nanowire probes for intracellular signaling of living cells

The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH₃ cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz.     

Sequence determinants of protein aggregation in human VH domains

Human antibody variable heavy (VH) domains tend to aggregate upon denaturation, for instance, by heat or acid. We have previously demonstrated that domains resisting protein aggregation can be selected from CDR-only repertoires by phage display. Here we analysed their sequences to identify determinants governing protein aggregation. We found that, while many different CDR sequences conferred aggregation-resistance, certain physico-chemical properties were strongly selected for. Thus, hydrophobicity and beta-sheet propensity were significantly lower among the selected domains, whereas net negative charge was increased. Our results provide guidelines for the design of human VH repertoires with reduced levels of protein aggregation.     

Contrasting disease and nondisease protein aggregation by molecular simulation

[Figurre: see text]. Protein aggregation can be defined as the sacrifice of stabilizing intrachain contacts of the functional state that are replaced with interchain contacts to form non-functional states. The resulting aggregate morphologies range from amorphous structures without long-range order typical of nondisease proteins involved in inclusion bodies to highly structured fibril assemblies typical of amyloid disease proteins. In this Account, we describe the development and application of computational models for the investigation of nondisease and disease protein aggregation as illustrated for the proteins L and G and the Alzheimer's Abeta systems. In each case, we validate the models against relevant experimental observables and then expand on the experimental window to better elucidate the link between molecular properties and aggregation outcomes. Our studies show that each class of protein exhibits distinct aggregation mechanisms that are dependent on protein sequence, protein concentration, and solution conditions. Nondisease proteins can have native structural elements in the denatured state ensemble or rapidly form early folding intermediates, which offers avenues of protection against aggregation even at relatively high concentrations. The possibility that early folding intermediates may be evolutionarily selected for their protective role against unwanted aggregation could be a useful strategy for reengineering sequences to slow aggregation and increase folding yield in industrial protein production. The observed oligomeric aggregates that we see for nondisease proteins L and G may represent the nuclei for larger aggregates, not just for large amorphous inclusion bodies, but potentially as the seeds of ordered fibrillar aggregates, since most nondisease proteins can form amyloid fibrils under conditions that destabilize the native state. By contrast, amyloidogenic protein sequences such as Abeta 1-40,42 and the familial Alzheimer's disease (FAD) mutants favor aggregation into ordered fibrils once the free-energy barrier for forming a critical nucleus is crossed. However, the structural characteristics and oligomer size of the soluble nucleation species have yet to be determined experimentally for any disease peptide sequence, and the molecular mechanism of polymerization that eventually delineates a mature fibril is unknown. This is in part due to the limited experimental access to very low peptide concentrations that are required to characterize these early aggregation events, providing an opportunity for theoretical studies to bridge the gap between the monomer and fibril end points and to develop testable hypotheses. Our model shows that Abeta 1-40 requires as few as 6-10 monomer chains (depending on sequence) to begin manifesting the cross-beta order that is a signature of formation of amyloid filaments or fibrils assessed in dye-binding kinetic assays. The richness of the oligomeric structures and viable filament and fibril polymorphs that we observe may offer structural clues to disease virulence variations that are seen for the WT and hereditary mutants.