hmmm... figure 8 results seem to need more analysis than that...

Phosphorylated HY5 is stable in the dark and not active!

Early figures showing the unphosphorylated HY5 has increased abundance-- performed in the DARK. The spaQ and mSPA1 (non-functioning spa1) mutants show this BUT (TEXT OPPOSITE FIG3) phosphorylated HY5 shows increased stability in the light-dark transition- degraded less quickly (fig6) In fig.3b- WT shows low HY5 level--- in the dark and long hypocotyls due to shade-avoidance elongation---- this is due to phospho-induced degradation of HY5 which would inhibit this response

EXPLAIN high ABUNDANCE? COP1 SUPPRESSOR 1 (CSU1), CSU2, PHYTOCHROME INTERACTING 1 (PIF1), and SIZ1 act as negative regulators of COP1-SPA complex to ultimately maintain HY5 homeostasis in etiolated seedlings TO ENOUGH FAST ABILITY TO REPRESS ELONGATION WHEN LIGHT RETURNS

SO theory that phosphorylated HY5 accumulates in the dark, despite having low activity---- in order to be able to quickly induce the photomorphogenesis response upon light exposure The unphosphorylated HY5 is more active in the light (results showing this?) AND is more degraded than phospho-HY5--- why more degradation--- to act as a regulatory mechanism so that other mechanisms are not OVERLY repressed by HY5--- need example--- lecture?

similar hypocotyl lengths for all conditions=== in far red light- the activity of HY5 is either not involved or not affected by phosphorylation---- is degradation inhibited under far-red--- COP1 degradation meaning no Ub? but what about the effect of phos on HY5 binding? this would suggest another TF is working hard under these conditions to inhibit photomorph.

far-red light found to induce repression of photomorphogenesis

HY5 upregulated under far-red https://doi.org/10.1093/plcell/koab254

HY5 supposed to repress expression

XTH15= increased expression null HY5 mutant- but not in the other mutants compared to WT

EXP2= sig decreased expression in S36A and sig increased in S36D--- suggesting increased and decreased repressive activity respectively

IAA19= INTERESTING RESULT!!! More expression in the HY5-overexpressing mutant than for plain WT--- this would suggest that HY5 activates expression? But the S36A mutant has sig lower expression than both WT and HY5. This result suggests that phospho-HY5 and normal overexpressing HY5 show the same activity- less repression of expression=====need investigation as to why--- anomaly?

SAUR36= similar to EXP2

CHS= opposite to EXP2= S36A overexpression causes increased CHS expression, and S36D reduces this expression compared to HY5-overexp.

KEY= there are differences in the level of effect of the S36D mutation compared to the non-overexpressing WT. Sometimes it has similar target expression level despite iits overexpression or it can have more or less functionality compared to WT. e.g. for CHS the expression is higher than for WT--- which suggests that the phosphorylation does not completely prevent induction of expression

ISSUE-- comparison of WT (not changed) to the HY5-S26D and S36A--- which are overexpressing lines... Shouldn't it be comparison of 'HY5' (overexpressing plain HY5) and the overexpressing mutants? THE WORDING IS UNCLEAR!!

WT= plain plant 'HY5'= HY5-overexpressing plant

ACT2= internal control gene https://www.future-science.com/doi/10.2144/04376RV01

XTH15= AT4G14130= Xyloglucan endotransglucosylase/hydrolase protein 15= catalyses hydrolysis of xyloglucan--- this molecule plays a role in enabling the cell to change its shape in growth and differentiation zones and to retain its final shape after cell maturation.---- in growth

EXP2= EXP2 is a nutrient-permeable channel =expression of AtEXP2 is associated with seed germination and subject to GA and stress regulation (http://dx.doi.org/10.1371/journal.pone.0085208)

IAA19= AT3G15540= Auxin-Regulated Protein That Functions Together with the Transcriptional Activator NPH4/ARF7 to Regulate Differential Growth Responses of Hypocotyl and Formation of Lateral Roots. https://doi.org/10.1105%2Ftpc.018630

SAUR36= SAUR36, a SMALL AUXIN UP RNA Gene, Is Involved in the Promotion of Leaf Senescence in Arabidopsis= auxin-regulated= final phase of leaf development https://doi.org/10.1104%2Fpp.112.212787

CHS= AT5G13930= chalcone synthase (CHS), a key enzyme involved in the biosynthesis of flavonoids= Chalcone synthases, the most well known representatives of this family, provide the starting materials for a diverse set of metabolites (flavonoids) which have different and important roles in flowering plants, such as providing floral pigments, antibiotics, UV protectants and insect repellents (https://doi.org/10.1007%2Fs11101-011-9211-7)

ACT2 (AT3G18780)= unchanged binding level between WT and mutants--- it is used as an INTERNAL CONTROL. This expression is NOT expected to be controlled by HY5 and therefore is a loading control (of sorts) to ensure that the total amount of DNA from the ChIP can be compared between the different conditions- in this case the mutants!

XTH15= significantly increased binding for S36A, and sig decreased for S36D--- but not abolished- showing that affinity is affected but not abolished by phosphorylation

EXP2= The same as for XTH15 (different levels of significance)

IAA19= significantly increased binding for S36A, but NOT sig decreased binding for S36D compared to WT

SAUR36= same as for XTH15

CHS= same as above

SOOO phospho-mimick HY5 (S36D) showed decreased CHS expression==== less activation= less activity. no Phospho- HY5 (S36A) showed increased CHS expression==== more activation= more activity

SO-- the phospho-mimic is showing reduced activity to repress the transcription of these genes

SO- no phosphorylation of HY5= higher activity to repress expression of growth and photomorphogenesis genes

Conversion of RNA to DNA or cDNA? and then quantitative PCR

the difference between mean values

Ch-IP-qPCR--- description

Issue with this entire experiment? The anti-GFP antibody is assumed to always be bound to the HY5- not just be cleaved/free GFP. Has there been previous experiments to establish that all WT/mutant HY5-GFP is expressed well with minimal cleavage? Otherwise, this experiment could be measuring the relative expression/accumulation of the GFP only!!! This is especially important for the light condition where the protein levels are similar between conditions

After 12 hours, all 3 HY5 conditions reach the same relative HY5 amount- which would suggest that phosphorylation status does not effect degradation in the light- only in the dark. Therefore there must be another factor in the HY5 degradation-- is it the amount of COP1? or is it increased activity of a de-ubiquitinating enzyme? Need to do research- under light conditions to establish the difference in binding partners compared to the light- what de-Ub enzyme. THEN look at the accumulation of COP1/de-Ub enzymes in light and dark---- or the level of HY5 interaction (by COP-IP) after light/dark conditions

Significant differences after 10 hours between the amounts of the mutants and WT. Significantly more S36D and significantly less S36A

WT HY5 shows gradual degradation upon darkness HY5-S36A (no phospho) shows increased rate of HY5 degradation in darkness HY5-S36D (mimic) shows decreased degradation in dark.

This suggests that in darkness, phosphorylation improves the stability of HY5

Is it assumed that (D) is dark for 4 days?

Does these 'lines' have any significance?

HY5 should accumulate during this time

Really need to confirm what this is...

Negative control that no COP1 is detected by the anti-COP1 in the cop1-4 mutant- but it is in the rest. This confirms that affinity for COP1

PROTEASOME inhibitor- to prevent degradation upon COP1 interaction- necessary to harvest enough COP1-HY5 to measure on the CO-IP

The smearing is caused by different amounts of HY5 + Ub. The more ubiquitination of the HY5, the slower it runs through the gel. Each band (smeared across the lane) represents a HY5 molecule with a different amount of ubiquitination

THIS IS AN ASSUMPTION- the smearing is not definitely caused by the poly-UB- it can be caused by sample loading issues/aggregates/overloading of a sample. Further experiments are required for confirmation

Comparison of the upper and lower blots--- the coomassie stains show that the upper blot has more protein present, which causes the increased chemiluminescent band intensity

S36D phospho-mimicking substitution mutation--- why? Serine (S) is often mutated to glutamic acid (E) (sometimes aspartic acid(D)) to mimic phosphorylation of the serine residue. This is because at pH7 (neutral and close to that of plant cytosols/used for experimental buffers--- check methodology!!) these AAs are negatively charged-- therefore mimicking the negative charge of the phosphate group. Conversely, mutating serine (S) to alanine (A) prevents potential phosphorylation. A is similar is structure and size to S-therefore unlikely to affect the activity/trafficking/interaction of a protein but CANNOT be phosphorylated as only serine/threonine/tyrosine can be.

There you go- the red numbers indicate the band intensities used to differentiate between the binding interactions of the WT and HY5 mutants

HY5-S36D interacts with COP1 statistically significantly less than WT and S36A HY5?? There looks to be significant difference, but it is not marked with an asterisk... assumed as obvious or untested?

Chart shows the statistically significant differences between the interaction of the WT and HY5-S36A compared to that of HY5-S36D with COP1. HY5-S36D interacts with COP1 significantly LESS than the other two HY5 types

SO the lower band intensity is shown for the HY5-S36D mutant than for the WT and the HY5-S36A mutant. The S36D mutation acts as a phospho-mimic (see !!! for why). Therefore the phosphorylation appears to negatively affect interaction of HY5 with COP1

REMEMBER- the S36D is a phospho-mimic!!! So this shows that the WT (HY5) and unable-to-be-phosphorylated HY5 (S36A) have stronger band intensities- which means that they bind more readily to the SPA1 background. Phosphorylation (in this case mimicked) reduces the binding of HY5 to SPA1

Th probability that the results are due to chance is less than 5%

3x repeats used to calculate the average and SD

This shows the raw band data which is quantified using the band intensity. The relative band intensities for the anti-GST bands have compared in the bar charts- a/b and c/d. * showing statistically significant difference between band intensity been the different HY5 pull downs. To show the difference in binding affinity for COP1 FUTURE; use isothermal calorimetry/SPR to measure the kinetics between COP1 and the different HY5 conditions- to get actual changes in the binding affinity

So this blot has affinity for the MBP tag on COP1- it will therefore be attached at all time. The HY5 and HY5 mutants are being washed over this background (each row has 1x HY5 added). The MBP-only column is a negative control to show that the HY5 do not interact with the MBP tag on its own.

same tag used for cop1 and spa1

So these results are supposed to show that unphosphorylated HY5 preferentially interacts with COP1/SPA1--- the phosphorylation reduces this interaction

The lettering is interesting... I think it means that conditions with the same letters (e.g. all a) are NOT statistically different from each other- whilst all letters that are different are SO- a conditions show statistically different hypocotyl lengths to b, c, d, and e conditions.

So HY5 should be active in the light- to prevent the elongation response/enhance photomorph. HY5 is more stable when phosphorylated- in the dark shows more accumulation. BUT is the activity reduced? This would suggest that under light conditions unphosphorylated BOTH has high activity and is MORE degraded by COP1/SPA1 complex. Therefore the phosphorylation acts to both regulated HY5 activity and abundance. why...

HY5-36A-GFP shows less hy5 in the B (boiled CIP) than in the CIP condition--- does this show more phosphorylation under CIP phosphatase conditions??

TAP-SPA1--- is this a control? Paik et al. 2019 paper uses both-- need to check if used for different things? Is this to show that the LUC tag is not having an effect in the complementation mutants? By showing that the phenotype is unaffected by the addition of the TAP-SPA1? BUT shouldn't they have done a LUC-SPA1 only expressing plant (with no spaQ mutant background) as well, to show this? That TAP-SPA1 and LUC-SPA1 produce the same phenotype?

paper that uses both TAP and LUC tagged SPA- not sure why you would use either?

1. no HY5 mutant= similar to WT-- showing that hy5 doesn't activate the elongation response in darkness-grown (etiolated) plants. as known it functions to inhibit elongation in light conditions- as it is unnecessary w/o shade.
2. spa124 mutant- largely reduced elongation response- but 1x spa protein is still expressed. This means that partial functioning of the shade-avoidance response is occurring with just spa3 present (compared to spaQ)
3. spaQ mutant- abolished elongation in response to darkness. Showing the importance of SPAs for allowing this response.
4. cop1-4 mutant- same phenotype as for spaQ, showing that they work in the same mechanism for activation/preventing inhibition of this response (by causing HY5 degradation).
5. LUC-SPA1/spaQ- LUC-SPA1 introduced to the quadruple spa mutant- to complement the lack of spa expressed (now expressing just spa1). Showed the same semi-response as for spa124- that also only expresses 1x spa kinase (spa3).
6. LUC-mSPA1/spaQ- the mutated mSPA1 is added to complement the spaQ mutant- but does not return the phenotype to that of the WT plant OR that of the spa124. Still no elongation in darkness- unlike for the SPA1 complementation. Showing that the mutated SPA1 does not function properly.
7. TAP-SPA1= plants expressing 35S promoter–driven N-terminal TAP (for tandem affinity purification)–tagged full-length SPA1--- why? search for tandem (mentioned once)-- look at paper using this? comparison to LUC-SPA?

TAP-SPA1

HY5 accumulation for the mSPA mutants and for spaQ--- thicker bands than for the LUC-SPA1/spaQ which has 1x SPA complementing the quadruple SPA mutant. this shows reduced degradation without the specific residue S36 for phosphorylation- leading to less HY5 degradation

Negative control for no CIP activity

So the Phospho-tagged SDS-PAGE gel will enhance the difference in mobility of phosphorylated---- or not. The lack of band shift is shown between the B and CIP conditions in fig.2c. This is the same result as shown for the HY5-S26A, so the phosphorylation is at this residue by the SPA kinase

So the CIP (phosphatase) treatment will remove the phosphorylation - therefore you would expect to the the mobility shift from higher on the gel (+P) to lower on the gel (no P)--- this is shown in figure2b (left side no SPA mutation).

This suggests that without the SPAs present, there was no mobility shift as there was no phosphorylation of the HY5

spaq= SPA quadruple mutant (1/2/3/4) with or without a luciferase added mSPA1= mutation R517E in kinase domain- significantly lower phosphorylation of HY5

Removal of phosphorylation from HY5

Yes, the radioassay does suggest reduced phosphorylation of the HY5.

The multiple bands again- multiple HY5 phospho-sites?

Need to look at the evidence for that

There is still faint bands for the mutant HY5- which could suggest another phosphorylation site for HY5. However, the intensity is similar to that of the SPA1 sample- which could just be showing radiolabelled-ATP bound to SPA1? But the band is slightly higher than both WT-HY5 and for SPA1- difference in mobility? or just gel error margin?

Also, there is a faint band for SPA1 in the negative control for +HY5 and -SPA1- there should be no band here. Is this transfer of sample between lanes of the gel, or an issue with the HY5 sample purity?

Torn gel? Should be re-done.

There is a possible mobility shift of the HY5 in the coomassie- which in addition to the radiolabelling indicates phosphorylation. But there are more than 2 bands- could this indicate more than one phosphorylation site? Again this links to the lack of mass spec coverage! BOTH HY5 and SPA1 are purified, so this is either P-HY% or a contaminating protein.

HOWEVER, this mobility shift is not seen in a) for the HY5. Could this be to do with lack of time for phosphorylation? Look in methods for timings? Or protein degradation occuring?

Obviously, more phosphorylation occurs over time- there is always a time-dependence to any reaction... Multiple mobility shifts, multiple phospho-sites?

Excel sheet showing the identification of a phosphorylation at s-36, and a M-O oxidation site closer to the C-terminal. HOWEVER, the mass spec coverage is not the full protein sequence, does this mean that they could've missed a phospho-site? are they certain that HY5 is mono-phosphorylated?

Is this what is being shown? Yes there is more phosphorylation at a higher concentration of SPA1- this is due to the limitation of the stoichiometry for SPA1 to phosphorylate. Should a serial dilution from very low to high of SPA1 be used to show extinguished phosphorylation at low SPA1 concentrations?

Not a huge difference between the amount of SPA1 in the + and ++ conditions?

So the ATP is radio-labelled- and will show up on the radio assay if the HY5 is phosphorylated.

The standard in vitro kinase radio assay involves immunocapture of the kinase of interest from cell lysate to a solid-phase support, typically using antibody-coated beads. After washing away other kinases and proteins, 32P-ATP and a peptide substrate with specificity to the kinase of interest are incubated with the captured kinase to allow kinase-catalyzed transfer of radio-labeled phosphate from the ATP to the substrate. The peptide substrate (both 32P-labeled and unlabeled) is captured using an affinity resin (e.g. biotin-conjugated peptides captured with a streptavidin solid support membrane) and unincorporated radioisotope (as 32P-ATP) is washed away. To assess kinase activity, the amount of radio-labeled phosphate incorporated into the substrate is quantitatively measured, typically using liquid scintillation.

In autoradiography dried polyacrylamide gels containing radiolabelled proteins are placed in direct contact with the appropriate X-ray film (e.g. Kodak X-Omat AR, Kodak SB-5, Kodak BMS2, Kodal BMR2, Fuji RX) where radioactive emissions (β-particles and/or β/γ-radiation) react with the silver halides in the film emulsion, resulting in the formation of elemental silver atoms which are visualized following photographic development of the films. [14C]-, [35S]-, [32P]-, [125I]- and [131I]-labelled proteins are readily detected using direct autoradiography while [3H]-labelled proteins are very weakly detected due to severe quenching of their low energy β-emissions by the polyacrylamide gel matrix.

Anti-tubulin controls- why lower intensity bands in light conditions. Due to loading of higher amounts in dark conditions

Are the additional bands from the cleaved GFP tag? Again, a WB against HY5, SPA, and GFP may elucidate these bands.

Really inconsistent controls!! Should have a HY5 and SPA2/3/4 control for each one!!

There are some strange bands presnt in the lanes, could a western blot for the HY5 and SPA proteins improve the certainty of what these bands are? HY5/degraded SPA/degraded, or something else?

is there some phosphorylation without the SPA2 present. radio-band + mobility shift in the HY5?

SPA2 band not really visible on the gel- why?

metabolic vs proteomic data- which is more valuable? Both show different parts (paper 1) proteomics shows the changes in expression of proteins, which is useful to know more about immune response. Metabolomics gives detail about the reactions occuring, the response to the bacteria, and the effect of the bacteria on the proteins present.

Disadvantage? both are difficult to apply to clinical samples. Can't use LC-MS for cows in all the farms- this is too expensive and takes too long. Much further research is required to apply the 'protein markers' identified to actually diagnosing and treating cows with mastitis. Lateral flow testing? Cows and milk production are economically important, so production of these types of tests may be viable?

Proteomics vs metabolomics- Proteomics utilises the genomic data to identify the different proteins that are identified- this assumes that they are WHOLE proteins that have not been highyl altered. This doesn't work for metabolites which are not genetically-encoded= they are a result of digestion/aleteration by other molecules/mechanisms. Therefore the genome will not show the range of different metabolites that are formed by metabolic pathways- but will show the original moleules (start point) just not the results of the reactions.

Compared to the proteomics data- reverse-phase chromatography sperates based on interaction with the hydrocarbon chains- not great for seperating metabolites (a mixture of small molecules) Metabolomics uses a different (hydrophobic) type of chromatography- that is good at seperating out lots of low abundance and similar molecules.

Lactate indicates the increased digestion of lactose in the milk by the bacteria- a key symptom of infection is the watery milk- due to lactose digestion? This is why distinguishing between the leucine and isoleucine is important- to show the digestion

MS is more sensitive- why? The separation of the samples by LC improves the ability to detect low abundance proteins within the samples. Whereas NMR (like MS too) is biased towards the high abundance proteins- disadvantage of NMR in metabolomics where the point is to detect all present proteins. Dis of MS only mass-based data- cannot distinguish easily between isomers? Whereas NMR gives structural information that allows this to be distinguished. In this paper the leucine vs. isoleucine components are unable to be distinguished. SEE OTHER LABEL TO SEE WHERE

Strength of response relative to the peak response. White= n/d? or what?

This may have affected the extraction of the metabolites?

Peak in the acid concentrations at 81h. Corresponds with the proteomics findings- the FXR pathway was activated by the mastitis infection.

Key changes in the metabolic pathway activities occuring at different times post-infections

KEGG database containing lots of data about metabolomic pathways in different organisms

0h and early hours show very similar responses between cows. This changes as the infection progresses- the difference in metabolomics increases over time.

Total # of proteins quantified- there will be many more that were not statistically significantly detected in the different cows. Change in the statistical threshold may identify more or less proteins- 2/6 samples? is this significant? This is where a larger sample size would be useful! However, due to the high workload required for proteomic analysis, this is difficult, expensive, and time-consuming to achieve.

Future experiments: look at the proteomes of cows at earlier time in the infection- to find an early prognostic marker for mastitis

Advantage/dis-- chemical change to protein- may affect positively/negatively the ionisability and detectability of the individual peptides-. Labelling- multi-plexing means that only one LC-MS run is required rather than multiple. This is good due to the lengthy amount of time required for the experiments and the lack of reproducibility of MS data- which makes comparison between the multiple datasets (at different phases) difficult

Did not show the same pattern of infection and recovery as for the other cows? Outlier in most of the data. fell ill more gradually and recovered more gradually- not known why. Should exclude this cow? No the aberrant cow shows consistently different proteomics, metabolomics, as seen in the next paper.

Acute-phase proteins (APPs) are a class of proteins whose concentrations in blood plasma either increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation. This response is called the acute-phase reaction (also called acute-phase response). The acute-phase reaction characteristically involves fever, acceleration of peripheral leukocytes, circulating neutrophils and their precursors.[1] The terms acute-phase protein and acute-phase reactant (APR) are often used synonymously, although some APRs are (strictly speaking) polypeptides rather than proteins

Key use of mutation screens for examining where a protein is in a pathway- if it is upstream, the phenotype will NOT be restored. So a null mutant has a phenotype, and a suspected member of the same pathway is deleted. If upstream- the phenotype remains the same- as this will not restore the pathway. If downstream- the phenotype may return to normal? Depends on the function of the original mutant in switching on or off the pathway.. If it functions to switch off the pathway, the second mutation should change the phenotype (as the constitutively one gene w/o the switch off will now be gone). And if functions to switch on- there should be no change. ???? I hate this so much.

So a loss of function phenotype whereby the lack of a gene causes no control between G2 phase and mitosis. Smaller cells= less time in G2= less growth= premature mitosis