Sequence-Based Prediction of Cysteine Reactivity Using Machine Learning

As one of the most intrinsically reactive amino acids, cysteine carries a variety of important biochemical functions, including catalysis and redox regulation. Discovery and characterization of cysteines with heightened reactivity will help annotate protein functions. Chemical proteomic methods have been used to quantitatively profile cysteine reactivity in native proteomes, showing a strong correlation between the chemical reactivity of a cysteine and its functionality; however, the relationship between the cysteine reactivity and its local sequence has not yet been systematically explored. Herein, we report a machine learning method, sbPCR (sequence-based prediction of cysteine reactivity), which combines the basic local alignment search tool, truncated composition of <i>k</i>-spaced amino acid pair analysis, and support vector machine to predict cysteines with hyper-reactivity based on only local sequence features. Using a benchmark set compiled from hyper-reactive cysteines in human proteomes, our method can achieve a prediction accuracy of 98%, a precision of 95%, and a recall ratio of 89%. We utilized these governing features of local sequence motifs to expand the prediction to potential hyper-reactive cysteines in other proteomes deposited in the UniProt database. We validated our predictions in <i>Escherichia coli</i> by activity-based protein profiling and discovered a hyper-reactive cysteine from a functionally uncharacterized protein, YecH. Biochemical analysis suggests that the hyper-reactive cysteine might be involved in metal binding. Our computational method provides a large inventory of potential hyper-reactive cysteines in proteomes and is highly complementary to other experimental approaches to guide systematic annotation of protein functions in the postgenome era

White-Light-Exciting, Layer-by-Layer-Assembled ZnCdHgSe Quantum Dots/Polymerized Ionic Liquid Hybrid Film for Highly Sensitive Photoelectrochemical Immunosensing of Neuron Specific Enolase

ZnCdHgSe quantum dots (QDs) functionalized with <i>N</i>-acetyl-l-cysteine were synthesized and characterized. Through layer-by-layer assembling, the ZnCdHgSe QDs was integrated with a polymerized 1-decyl-3-[3-pyrrole-1-yl-propyl]­imidazolium tetrafluoroborate (PDPIT) ionic liquid film modified indium tin oxide (ITO) electrode to fabricated a photoelectrochemical interface for the immobilization of rabbit antihuman neuron specific enolase (anti-NSE). After being treated with glutaraldehyde vapor and bovine serum albumin successively, an anti-NSE/ZnCdHgSe QDs/PDPIT/ITO sensing platform was established. Simplely using a white-light LED as an excitation source, the immunoassay of neuron specific enolase (NSE) was achieved through monitoring the photocurrent variation. The polymerized ionic liquid film was demonstrated to be an important element to enhance the photocurrent response of ZnCdHgSe QDs. The anti-NSE/ZnCdHgSe QDs/PDPIT/ITO based immunosensor presents excellent performances in neuron specific enolase determination. The photocurrent variation before and after being interacted with NSE exhibits a good linear relationship with the logarithm of its concentration (log <i>c</i>_NSE) in the range from 1.0 pg mL^–1 to 100 ng mL^–1. The limit of detection of this immunosensor is able to reach 0.2 pg mL^–1 (<i>S</i>/<i>N</i> = 3). The determination of NSE in clinical human sera was also demonstrated using anti-NSE/ZnCdHgSe QDs/PDPIT/ITO electrode. The results were found comparable with those obtained by using enzyme-linked immunosorbent assay method

Tetraspanin-enriched HIV-1 positive endosomal compartments in infected MDMs are not accessible to the external environment.

<p>Human MDMs were infected with VSV-G-pseudotyped 29/31 KE mutant HIV-1. 8 days post infection, cells were fixed and immunolabeled for tetraspanins (red, anti-CD81, anti-CD9, or anti-CD63) without cell permeabilization, followed by permeabilization and immunolabeling for HIV-1 Gag (green, anti-MA). Image acquisition was performed with an Improvision/Perkin Elmer spinning disc confocal fluorescence microscope. Bars represent 16 µm.</p

Endosomal compartment markers colocalize with Gag in macrophages infected with 29/31 KE endosomal-targeting mutant virus after permeabilization.

<p>Human MDMs were infected with VSV-G-pseudotyped 29/31 KE mutant HIV-1. 8 days post infection, cells were fixed and immunolabeled for HIV-1 Gag (green, anti-MA) and tetraspanins (red, anti-CD81, anti-CD9, or anti-CD63) after cell permeabilization, followed by imaging acquisition with an Improvision/Perkin Elmer spinning disc confocal fluorescence microscope. Bars represent 16 µm.</p

Low molecular weight dextran is largely excluded from VCCs in macrophages.

<p>HIV-infected MDMs were incubated with Texas red dextran, 3000 MW, at 37°C or 4°C for 30 minutes. Cells were then fixed and stained for Gag (green). (A-B) Representative images of cells incubated at 37°C. (C) Representative image of cells incubated at 4°C. (D) Representative image of cells incubated at 4°C with no wash prior to fixation. Bars  =  11 µm. (E) Quantitation of colocalized voxels from 3D image stacks derived from ten cells at each temperature, presented as µm³. Error bars indicate standard deviation.</p

Quantitation of colocalization of Gag in macrophages infected with WT or 29/31 KE endosomal-targeting mutant virus before and after permeabilization.

<p>Volocity software package (Perkin Elmer) was used to quantify colocalized pixels before (grey) and after (white) permeabilization of cells. Error bars represent standard deviation, from a total of 30 cells examined for each experiment.</p

The majority of virus-containing compartments in infected MDMs are inaccessible to a cell surface label.

<p>(A-B) Uninfected Human MDMs cultured on ACLAR embedding film were fixed and stained with cationized ferritin (CF), followed by standard electron microscope processing procedures. Images were then obtained under a Hitachi H-7500 transmission electron microscope. Cationized ferritin labeled plasma membrane is seen along the plasma membrane (PM). (A) and (B) represent uninfected macrophages, bars  =  0.5 µm. IC  =  apparent intracellular space stained with CF. (C) HIV particles were seen at PM and stained with CF on periphery of cells (arrows). Bar  =  1 µm. (D) CF is seen staining HIV particles underlying a PM fold (arrow), while deeper VCCs lack CF staining. Bar  =  0.5 µm. (E-F) CF staining of membrane protrusions contrasts with lack of CF in intracellular VCCs (F is higher magnification view of boxed region in E). Bar  =  1.0 µm (E), 0.5 µm (F). (G-I) Additional views of PM staining with CF and exclusion of CF from VCC. (H represents higher magnification view of boxed region from G, bars  =  2 µm). (J) VCCs were counted as CF+ or CF- from 329 apparent intracellular VCCs in more than 50 cells. The number of CF-negative compartments vs. CF-positive compartments is indicated.</p

FIP1C depletion and HIV-1 Env incorporation in HeLa cells.

<p>(A) The efficiency of depletion of Rab11a-FIPs was measured by real time PCR (top). Infectivity of released particles following depletion of each FIP family member is shown below. (B) Depletion of FIP1C was confirmed at the protein level by immunoblotting with a FIP1C-specific antiserum. Env levels in both cell lysates and virions harvested from the FIP-depleted HeLa cells were assayed by immunoblotting. (C) Restoration of Env incorporation following shRNA-mediated depletion. Two constructs were used in this experiment: FIP1C cDNA is an shRNA sensitive GFP-tagged plasmid; while FIP1C* includes silent mutations in the shRNA target sequence rendering it shRNA-resistant. 2 ug and 4 ug of each FIP1C construct were used in the repletion assay as indicated at the top of the blot. (D) Infectivity of viral particles from the experiment shown in panel D as evaluated using TZM-bl indicator cells. Lanes are numbered and correspond to the blot above. Statistical comparisons utilized the unpaired t-test, * = p<.05; ** = p<.01.</p

The incorporation of Env onto HIV-1 particles is saturable in a cytoplasmic tail-dependent fashion.

<p>(A) Increasing amount of wildtype and the tailless mutant (CT144) of NL4-3 Env protein expressing plasmids were co-transfected with fixed amount of pNL4.3Env- proviral plasmid into HeLa cells. Progeny viral particles were harvested two days after transfection through a 20% sucrose cushion and the particles were lysed with SDS loading buffer. Proteins were analyzed by immunoblot using Env and CA specific antibodies. (B) The intensity of each Env band and Gag band in the particle immunoblot shown in A was quantified by Licor software. Background-subtracted pixel intensity values were plotted on the graph as Env band intensity versus CA band intensity. (C) The absolute quantity of Env and CA/P24 on particles was measured by gp120 ELISA and p24 ELISA. The number of Env molecules per particle was calculated as an approximation, assuming that 5000 CA molecules are present in one HIV-1 virion. (D) The infectivity of the progeny viral particles was measured using TZM-bl reporter cells. Infectivity is plotted as number of blue cells per nanogram of p24 antigen used in the infection assay.</p

Rab14 interaction with FIP1C is required for HIV-1 Env incorporation.

<p>(A) HeLa cells were transfected with proviral plasmid pNL4-3 or NL4-3 CT144 and either a dominant negative form of Rab14 (Rab14S25N) or a constitutively active form of Rab14 (Rab14Q70L). 48 hours after transfection, cellular and particle content of HIV-1 Env was examined by Western blot. Numbers above Env blots represent densitometry values with leftmost lanes set to 100. (B) shRNA-mediated depletion of Rab11 or Rab14 was performed in HeLa cells. Following selection in puromycin, cells were transfected with either pNL4-3 or pNL4-3 CT144 plasmid. Cellular and particle-associated Env was detected by immunoblotting 48 hours following transfection. (C) shRNA-resistant GFP-FIP1C* WT and GFP-FIP1C* (S580N/S582L) were transfected together with pNL4-3 in control HeLa cells or FIP1C-depleted HeLa cells as in the repletion experiment described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003278#ppat-1003278-g002" target="_blank">Figure 2</a>. Input FIP1C* plasmid levels were 2 µg in lanes 3, 5, 7, and 8 and increased to 4 µg in lanes 4 and 6. In a separate experiment the ability of Rab11 binding mutant GFP-FIP1C* I62E and GFP-FIP1C* D622N to rescue Env particle incorporation in FIP1C-depleted HeLa cells (lanes 7 and 8). Note that gp160 and gp120 were detected with polyclonal antiserum; gp41 particle blots were probed with a monoclonal specific for gp41.</p