Post translational modification of proteins

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Post-translational modification of proteins refers to the chemical alterations that occur to proteins after their synthesis, which can affect their function, stability, localization, and interactions. These modifications include processes such as phosphorylation, glycosylation, ubiquitination, and methylation, playing crucial roles in regulating cellular activities and signaling pathways.

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HYPOTHESIS AND THEORY Dysbiosis May Trigger Autoimmune Diseases via Inappropriate Post-Translational Modification of Host Proteins

by aaron lerner

The gut ecosystem with myriads of microorganisms and the high concentration of immune system cells can be considered as a separate organ on its own. The balanced interaction between the host and microbial cells has been shaped during the... more

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Tryptophan and Non-tryptophan Fluorescence of the eye Lens proteins provides Diagnostics of Cataract at the Molecular Level

by Dmitry Gakamsky and

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications... more

Figure S3h. Fluorescence lifetime measurements of Kyn 500nm (left) and its residue in Ac-AA-Kyn- AA-NH, at 540nm upon 372nm excitation (right). The data were fitted by a double- exponential models, Kyn (t;=34 +15ps (95.4%) and 1.=0.97ns (4.6%), average lifetime 60ps), and Ac-AA-Kyn-AA- NH, (t;= 4.6ns (34.6%) and t,=8.2nsns (65.4%), average lifetime 6.9ns).

Figure S3f. Fluorescence lifetime measurements of NFK at 420 nm upon 317 nm excitation (left) and its residue in Ac-AA-NFK-AA-NH, at 420 nm upon 372 nm excitation (right). The data were fitted by a double-exponential model (t; = 43 ps (95.4%) and 12 = 2.5 ns (4.6%), average lifetime 0.17 ns) ora 3-exponential model (t,=0.6 ns (35.5), t=2.4 ns (52.8%) and 13 = 6.4 ns (11.7%), average lifetime 2.23 ns) respectively.

Figure S3a. Fluorescence excitation (blue) and emission (green) spectra and lifetime measurements (inset) of NATA in PBS. The fluorescence time-response was measured at 280 nm upon 350 nm excitation and fitted by a mono-exponential model (t = 2.9 ns).

Figure S3b. Fluorescence excitation (blue) and emission (green) spectra and lifetime measurements (inset) of Ac-AAWAA-NH) peptide in PBS. The fluorescence time-response was measured at 350 nm upon 280 nm excitation and fitted by a double-exponential model (t; = 1.1 ns (33.4%) and t2 = 2.0ns (66.6%); average lifetime t = 1.7 ns).

Figure 1. (A) Excitation of eye lens fluorescence in a normal (left) and UV-irradiated porcine lens by a 317 nm- LED. (B) A zoomed trace of the 317 nm-beam in the normal lens. (C) Analysis of the change in the fluorescence intensity of the excitation beam.

Figure 2. Emission spectra of a UV-irradiated (A) and normal porcine eye lens (B) measured at 305 nm (blue), 317nm (green), 325 nm (red) 370 nm (cyan) excitation wavelengths. The respective normalised spectra of the normal and UV-irradiated lenses are shown in the Insets. Emission spectrum map of solubilised porcine lens after UV irradiation (C) and the insoluble fraction of an emulsified donor sample (NC++) (D) upon 300 nm (blue), 317 nm (green), 325 nm (red) 370 nm (cyan), 400 nm (magenta), 450 nm (yellow) and 500 nm (black) excitation wavelengths. Normalised spectra of the UV-irradiated and the emulsified lens (Insets).

Figure 3. Fluorescence spectral and lifetime parameters of Trp, its derivatives and ArpP (A). A simulated emission spectrum generated from emission spectra of Trp and the OH-Trp, ArgP, NFK, Kyn and OH-Kyn in the 5:37:11:32:11:5 proportion (B) and spectral dependence of average fluorescence lifetime (inset). A typical example of fluorescence lifetime responses of the insoluble fraction of an emulsified donor eye lens (NC++) measured across the emission spectrum in the 350-550 nm range upon 317 nm excitation (C). Steady-state emission spectrum of this sample upon 317 nm excitation (D) and a spectral dependence of averaged lifetime calculated from the data shown in the left panel (inset).

Figure 4. Absorption spectra of AAWAA before (black) and after (red) UV irradiation (A). Emission spectra of AAWAA before UV irradiation upon 280 nm excitation (dash black), and after UV irradiation upon 280 nm (blue), 300 nm (green) 320 nm (red), 350 nm (cyan), 400 nm (magenta), 450 nm (yellow) and 500 nm (black) excitation (B). Normalised emission spectra (inset). Decomposition of the emission spectrum of Fr5 measured upon 372 nm excitation (black) using spectra associated with 7,7, and 7; (blue circles) and 7, (red circles) lifetime components. The parameters were calculated by evaluating fluorescence time-responses of Fr5 in the 400-600 nm range by a 4-exponential model with “linked” lifetime parameters. (C) Blue-shifted by 5nm fluorescence spectra of NFK (blue) and fluorescence spectrum of Kyn (red) in PBS.

Figure 5. Fluorescence spectra of the soluble (A) and insoluble (B) fractions of emulsified donor eye lens proteins measures upon 310 nm excitation. The spectra were normalised on the emission spectrum of Trp (black) measured in samples of porcine eye lens proteins solubilised either in PBS (left panel, black) or in PBS/7 M urea buffer (right panel, black) upon 310 nm excitation. (C) Decomposition of the emission spectrun (excitation 310 nm) of the insoluble fraction of an emulsified cataractous sample (NC++) (black) over fluorescence spectra of NATA (1), AA-OH-Trp-AA (2), ArgP (3), AA-Kyn-AA (4), AA-Kyn-AA (5) and AA- OH-Kyn-AA (6) (blue) and corresponding fit-function parameters: C, = 0.84, C, = 0.21, C;=0.20, C,=0.46, C; = 0.05, Cs = 0.08. Residuals function (bottom panel). The spectral decomposition was carried out using equation (4). Correlation of cataract grade with the normalised cumulative fraction (C, + C+ C,)/C, for 21 emulsified eye lens samples (D).

Figure S3d. Fluorescence excitation (blue) and emission (green) spectra and lifetime measurements (inset) of Ac-AA-OH-Trp-AA-NH), in PBS. The fluorescence time-response was measured at 380 nm upon 317 nm excitation and fitted by a double-exponential model (t, = 0.6 ns (4.8%) and 12 = 4.7 ns (95.2%); average lifetime t =4.5 ns).

Figure S3e. Fluorescence excitation (blue) and emission (green) spectra of NFK in PBS

Figure 6. Decomposition of emission spectra (excitation 317 nm) of a 30 (A) and 80 years old (B) donor lenses over fluorescence spectra of NATA (1), AA-OH-Trp-AA (2), ArgP (3), AA-Kyn-AA (4), AA-Kyn-AA (5n) and AA-OH-Kyn-AA (6). Fit-functions (blue) with C, = 0.74, C,=0.14 C;=0.14, C,= 0.84, C; = 0.03 C;=0.12 (panel A) and C, =0.42, C,=0.12, C;=0.39, C,=0.72, C; =0.00, C; = 0.09 (panel B). Residuals functions (A,B bottom panels). The spectral decompositions were carried out using equation. (4). Correlation of age with normalised cumulative fraction (C, + C3 + C,)/C, for 14 donor lenses with age related nuclear cataracts (C).

Figure $1. Size exclusion chromatograms of a control (blue) and UV-irradiated solubilised porcine eye lens samples (green) performed at 0.5ml/min flow rate in a Superdex 200 column (GE Healthcare). Inset: Size-exclusion column (Superdex 200) calibration curve. The data were obtained by applying samples of Aprotinin (6.5 kD; 21.21 min), Ribonuclease (13.7 kD; 19.68 min), Carbonic Anhydrase (29 kD; 17.86 min), Ovalbumin (44 kD; 15.50 min), Conalbumin (75 kD; 13.35 min), Aldolase (158 kD; 111.1 min) and Ferritin (440 kD; 8.91 min) in PBA to the column. The data were fitted by Sth degree polynomial function y([D])= -10.557x° + 899.14x* - 30478x° + 5.1466e+005x° - 4.3436e+006x + 1.4768e+007, ([x] min). ‘Edinburgh Instruments, 2 Bain Square, Livingston, EH54 7DQ, UK; “Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 6 UK; Edinburgh Eye Pavilion, Chalmers Street, Edinburgh EH3 9HA, UK; ‘Lein Applied Diagnostics, Reading Enterprise Centre, Whiteknights Rd, Reading RG6 6BU, UK

Figure S3j. Fluorescence lifetime measurements of OH-Kyn at 540 nm (left) and its residue in Ac- AA-OH-Kyn-AA-NH) at 570 nm (right) upon 442 nm excitation. The data were fitted by 3- exponential models: OH-Kyn (t; = 0.1 ns (84.1%), t2 = 0.8 ns (12.1%) and t3= 4.4 ns (3.8%), average lifetime 0.35ns); Ac-AA-OH-Kyn-AA-NH, (t,;= 0.8 ns (33.4%), t2= 3.4 ns (44.3%) and 73= 11.7 ns (22.3%), average lifetime 4.4ns).

Figure S3l. Fluorescence excitation (blue) and emission (green) spectra and lifetime measurements (inset) of Pentosidine in PBS. The fluorescence time-response was measured at 370 nm upon 315nm excitation and fitted by a single-exponential model (t = 4.0 ns).

Figure S4. Global analysis of fluorescence time-responses (left panel) measured in the insoluble fraction of a NC++ sample upon 317nm excitation in the 350 — 550nm range. The analysis was carried out by the FAST software (Edinburgh Instruments, UK) by a 4-exponentil model with “linked” lifetime parameters. Graphical representation of the lifetime components (right top panel). Spectra associated with the lifetime components (right bottom panel).

Fluorescence lifetime measurements have been employed to decompose this spectrum into its distinct components. To this end we measured fluorescence time-responses along the spectrum in the 400 — 600 nm range upon 372 nm picosecond excitation. It was found that the emission time-responses on the red slope (Fig. 4D, red) were bi-exponential with major 7.8ns (71.1%) and minor 4.1ns (28.9%) lifetime components (inset, red). Based on spectral position this was attributed to emission of AA- Kyn-AA. The emission response on the blue slope (Fig. S7, left panel, blue) containing 0.8ns (52.5%), 2.8ns (43%) and 7.0ns (4.5%) lifetime components (inset, blue) is likely to be predominantly caused by emission of AA-NFK-AA. Fig. S7. Fluorescence time-responses of Fr5 measured at 410nm (blue) and 540nm (red) upon 372nm excitation and Instrument Response Function (black) (left panel). Graphical representation of the model parameters for the 410nm (blue) and 540nm (red) time-responses (inset). Decomposition of the emission spectrum of Fr5 measured upon 372nm excitation (black) using spectra associated with T,,t2 and 13 (blue circles) and ty (red circles) lifetime components. The parameters were calculated by evaluating fluorescence time-responses of Fr5 in the 400 - 600nm range by a 4-exponential model with “linked” lifetime parameters. Blue-shifted by 5nm fluorescence spectra of NFK (blue) and fluorescence spectrum of Kyn (red) in PBS (right panel).

Fig. S8. Global analysis of fluorescence time-responses (left panel) measured in the HPLC Fr5 containing a mixture of Ac-AA-NFK-AA-NH, and Ac-AA-Kyn-AA-NH, peptides upon 372nm excitation (vertical polarization) in the 350 — 550nm range at the magic angle. The analysis was carried out by the FAST software by 4-exponentil model with “linked” lifetime parameters. | Graphical representation of the lifetime components (right top panel). Spectra associated with the first three (0.22ns, 1.06 and 3.07ns) (blue) and the forth (7.64ns) (red) lifetime components (right bottom panel).

Table 1. PTM rates in crystallins of a human emulsified lens with nuclear cataract (NC++).

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