BACKGROUND
proteolytic processes are of vital importance due to the irreversibility of peptide bond cleavage.
spatio-temporal regulation of proteolysis is therefore of highest significance in determining the fate of cells.
to date more than  <dig>  gene sequences have been annotated as coding for proteolytic enzymes.
the diversity of proteases is further reflected in broad tissue-, compartment- and substrate specificities.
most of all protease genes encode for serine peptidases , followed by metallo  and cysteine peptidases   <cit> .
because of this variety, one key issue of degradomics is to investigate networks of proteases in order to explain distinct proteolytic functions in the spatio-temporal context of substrate cleavage  <cit> .
it is common practice to employ proteolytic activity assays to achieve this goal.
in this report we focused on cysteine cathepsins b, k, l and s, and analyzed their processing patterns using a natural substrate, thyroglobulin , while taking into account the suspected redox- and ph values of the physiological cleavage environments.
it has been shown before that the cysteine cathepsins b, k, l and s are localized to the endo-lysosomal system of thyroid epithelial cells.
moreover, they are present at extracellular locations such as the apical plasma membrane and within the follicular lumen of the thyroid gland  <cit> .
generally, cysteine cathepsins of the papain superfamily are well known to engage in bulk protein turnover at the increasingly acidic ph values of endocytic compartments  <cit> .
nevertheless, the proteolytic potency of cysteine cathepsins towards peptidic or natural substrates has been demonstrated to expand over a wide range including neutral ph values  <cit> .
in fact, cysteine cathepsins were often accredited as extracellularly acting enzymes in vivo .
diverse protocols are available that describe methods to perform activity assays of cysteine cathepsins at neutral conditions, usually after activation of the proteases by incubation with reducing agents  <cit> .
in reality, however, the extracellular space is not only a neutral but also an oxidizing environment, as much as endosomes and lysosomes exhibit not only acidic but also reducing conditions.
in this study, we have chosen redox-values of - <dig> mv to mimic the reducing environment of the cysteine-rich endosomes and lysosomes  <cit> .
moreover, endo-lysosomes are characterized by increasingly acidic conditions  <cit> , and we considered ph  <dig>  and ph  <dig>  as closely reflecting the endosomal and lysosomal milieu, respectively.
on the other hand, the thyroid follicle lumen represents a secluded compartment in which the tight junctions between thyroid epithelial cells tie up the extracellular storage space for tg from the intercellular spaces between thyrocytes  <cit> .
the follicle lumen is assumed to be neutral in ph thereby rendering tg proteolysis by extracellular hydrolases unlikely .
however, more recently we provided evidence for tg proteolysis mediated by cysteine cathepsins within the extracellular thyroid follicle lumen before its endocytosis  <cit> .
extracellularly stored tg of the human thyroid follicle lumen is covalently cross-linked by disulfide bonds  <cit>  which can be formed in a self-assisted fashion via the cxxc-boxes within tg  <cit> , thereby attesting an oxidizing milieu within the thyroid follicle lumen.
furthermore, the direct pericellular environment adjacent to the apical plasma membrane of thyroid epithelial cells is oxidizing due to the presence of an h2o <dig> generating system which enables thyroid peroxidase to iodinate tg at neutral ph  <cit> .
we have chosen a value of - <dig> mv to mimic the extracellular space which is slightly more oxidizing than the range of - <dig> mv to - <dig> mv that was reported for the lumen of the endoplasmic reticulum  <cit> .
as yet, there are only very few studies which consider both, ph values and redox potentials of the cleavage environment  <cit> .
the assay described herein therefore employs buffers accounting for the conditions in the follicle lumen equivalent to the extracellular space or within endo-lysosomal compartments.
we assessed the suitability of such buffers to promote proteolytic cleavage of tg by specific cysteine cathepsins in order to simulate proteolysis within thyroid follicles, i.e.
within an extracellular compartment, or within the endo-lysosomes of thyrocytes .
processing of tg in the thyroid serves as an excellent model system for our purposes, because it has been demonstrated that  the cysteine cathepsins b, k, l and s cleave tg in vitro  <cit> ,  tg is located in all compartments where cysteine cathepsins principally meet their substrates, i.e.
endosomes, lysosomes, and in the extracellular space ,  cysteine cathepsins and tg represent a naturally occurring and physiologically relevant enzyme-substrate couple in vivo  <cit> .
under physiological conditions, proteolysis of tg leading to thyroid hormone liberation is accomplished by cysteine cathepsins in a sequential process starting with the solubilization of tg from its covalently cross-linked macromolecular storage forms that fill the extracellular follicle lumen.
the initial substrate cleavage results in the production of smaller protein fragments that are internalized by thyroid epithelial cells together with partially processed tg  <cit> .
accordingly, liberation of the thyroid hormones t <dig> and t <dig>  which are essential for the regulation of mammalian development and metabolism begins extracellularly and is perpetuated within endocytic compartments of thyroid epithelial cells.
this study aims at elucidating crucial roles of selected cysteine cathepsins in this sequence of prohormone processing.
the development of an in vitro assay system mimicking the in situ ph and redox conditions enabled analysis of the potency of cathepsins b, k, l and s to cleave their natural substrate tg and to liberate thyroid hormones under conditions simulating favorable  versus non-favorable  compartments.
RESULTS
localization of cysteine cathepsins in the human thyroid gland
the subcellular localization of cysteine cathepsins has been studied in detail in almost all tissues but not to the same extent in the human thyroid gland.
therefore and in order to analyze the distribution patterns of particular cysteine cathepsins in the compartments of tg processing, human thyroid tissue was immunolabeled with antibodies against cathepsins b, k, l, and s in single and double labeling experiments.
cathepsins b, k, and l were co-localized within the same intracellular vesicles .
cathepsin s appeared to be also localized to distinct vesicular compartments, because co-localization with cathepsins k and l was not as prominent .
some cathepsin l-positive vesicles were even lacking cathepsin s .
likewise, cathepsin k-positive structures were often negative for cathepsin s .
consequently, cathepsin s-positive vesicles lacking cathepsins k or l were frequently detected .
moreover, all cathepsins analyzed were detected in the extracellular space, i.e.
within the follicle lumen, indicating their secretion .
note, that the extracellular follicle lumen  represents a storage compartment for tg, hence, all cathepsins are found in close proximity to their natural substrate already here.
cathepsin s was prominently present in the extracellular follicle lumen .
cathepsins k and l were additionally found pericellularly in close association with the apical plasma membrane , where thyroid hormone liberation by tg processing is believed to start.
we conclude that trafficking of proteases in thyroid epithelial cells results in a distinct protease composition within vesicles of the endocytic pathway in addition to their secretion into the extracellular space.
hence, it is important to analyze tg processing with different combinations of cysteine cathepsins and to simulate substrate cleavage at the conditions of the respective compartments, i.e.
extracellular follicle space, endosomes and lysosomes.
establishment of buffers with stable redox potentials at distinct ph values
buffers representative for the extracellular space, the endosomal compartment and for lysosomes were established through the use of a two-buffer system of citric acid, sodium phosphate, and l-cysteine.
in order to verify buffer stability, the actual ph and redox values were determined for a minimum time period of  <dig> hours.
the buffer simulating extracellular conditions with an oxidizing redox potential of - <dig> mv and neutral ph  <dig>  was shown to be stable over  <dig> hours .
although addition of tg marginally lowered redox and ph values, the overall stability under extracellular conditions was more stringent in the presence of substrate .
buffers mimicking endosomes and lysosomes were adjusted to - <dig> mv and differed from one another in their ph values, only.
the endosomal-representative buffer was set to ph  <dig>  and the lysosomal to ph  <dig> .
the stability of these buffers with respect to redox potential and ph value was verified over a  <dig> h time period  as these buffers were stable from the very beginning.
determination of cleavage sites within tg
isolated tg was incubated with cathepsins in buffers simulating extracellular conditions and the extent of substrate cleavage was compared to that achieved under conditions characteristic for intracellular proteolysis within endo-lysosomal compartments.
cathepsins b, k, l and s or combinations thereof were incubated with tg in the respective buffers and proteolysis was allowed to occur for  <dig> hours.
preparations were then heated in lds sample buffer without any further addition of reducing agents.
this procedure ensured consistent behavior of all samples from the same conditions upon separation by page since they were run on separate gels.
fragmentation patterns were analyzed by sds-page followed by immunoblotting using antibodies specific for tg  and by n-terminal sequencing of selected tg-fragments .
note, that for all incubations an identical amount of tg was subjected to cysteine cathepsin degradation, whereby the particular amount of distinct enzymes was kept constant in all incubations.
therefore, the ratios of total enzyme to substrate differed between single and combinatorial incubations .
we were especially decided about such conditions, because we consider this setup to best reflect tg cleavage in situ.
as we have shown in the localization studies , vesicles may contain only one type of enzyme or combinations of different enzymes.
for the extracellular follicle lumen, similar approximations might be relevant, since we observed almost no co-localization of the distinct cysteine cathepsins in the extracellular space, but different enzymes were detectable and can therefore, in principle, cleave luminal tg.
hence, the observed degradation patterns must be interpreted as resulting from different combinations of enzymes acting sequentially and/or at the same time on tg.
under neutral and oxidizing conditions mimicking the extracellular thyroid follicle lumen full-length tg of approximately  <dig> kda was cleaved by each of the cysteine cathepsins tested, even though these buffer conditions are considered as 'non-favorable' for proteolytic action of cysteine cathepsins .
the effectiveness of substrate-cleavage was obvious from the appearance of two fragments of approximately  <dig> and  <dig> kda , which were also detected when the assay buffers reflected more favorable endosomal cleavage conditions, i.e.
a reducing and slightly acidic environment .
in addition to the high molecular mass fragments, all samples containing cathepsin s displayed a specific  <dig> kda fragment under extracellular conditions .
the same-sized fragment was generated at conditions characteristic for endosomes and lysosomes by several combinations of cysteine cathepsins but notably not by cathepsin k alone or in combination with cathepsins l and s. the analysis of degradation patterns using assay conditions mimicking lysosomal cleavage revealed a number of low molecular mass fragments of tg as generated by all cathepsin combinations .
samples containing cathepsin l lacked the intact, full-length tg , but contained numerous smaller fragments of 40- <dig> kda .
note that all preparations contained equal amounts of tg at the start of incubation with cysteine cathepsins under different conditions.
the lanes were loaded after the incubation period with identically sized aliquots from each preparation.
hence, the absence of signal in certain lanes of the immunoblots is representative of extensive tg degradation rather than being the result of unequal loading of the gels.
accordingly, blank or weakly labeled lanes indicate complete degradation of tg by the respective cysteine cathepsin/s at these conditions.
as a control of its stability, tg was incubated in the respective buffers, but without any protease added .
in order to determine the cleavage sites that were used by cysteine cathepsins, characteristic tg fragments were n-terminally sequenced .
a band with a molecular mass of approximately  <dig> kda exhibited the same n-terminus as unprocessed tg in all instances, thus representing intact, full-length protein.
tg-fragments originating from proteolytic processing by cathepsin s under extracellular conditions had molecular masses of approximately  <dig> kda,  <dig> kda and  <dig> kda.
two of these fragments exhibited newly generated n-termini, while the  <dig> kda fragment showed the same n-terminus as unprocessed tg.
arrowheads indicate relative positions of identified cleavage sites as deduced from n-terminal sequencing of extracellular-  or endosomal-like tg degradation .
in figure 5d a scheme of lysosomal cleavage sites is depicted, which was determined by dunn and colleagues  <cit>  under near-physiological conditions since lysosomal extracts were used to degrade tg.
the schematic drawings in figure  <dig> illustrate that the cleavage sites varied within tg depending on the conditions under which cleavage took place.
analysis of physiological significance of tg cleavage
determination of cathepsin cleavage sites revealed that the n-terminal half of tg was prone to extracellular and endosomal cleavage, whereas the thyroid hormone-rich c-terminal half is, according to  <cit> , best cleaved under conditions of lysosomal tg processing.
in order to implement a physiologically relevant read-out for efficiency of substrate cleavage, we analyzed the in vitro-degradation samples for their content of liberated, free thyroxine  by elisa.
under extracellular conditions, ft <dig> was only detected in samples that contained cathepsin s alone or in combinations with other cathepsins .
under endosomal conditions, the overall efficiency of ft <dig> liberation was considerable, about  <dig> fold higher than compared to extracellular conditions with values of ft <dig> ranging from approximately  <dig> to  <dig> pm .
yet, another augmentation in the amount of liberated t <dig> was observed under conditions simulating lysosomes where cathepsins b, k and l performed equally well by liberating t <dig> in a range from  <dig> to  <dig> pm .
discussion
one important mechanism to control protease activity is given by endogenous inhibitors, another is represented by the dependency of proteolytic enzymes on the ph and redox potential of the cleavage environment.
whereas ph sensitivity has been studied quite extensively , much less is known about their sensitivity to the redox potential of the environment.
endo-lysosomal cysteine proteases like cathepsins l and b are considered highly instable at neutral ph values with half lives of  <dig>  and  <dig> minutes, respectively  <cit> .
the inactivation appears to be irreversible and is associated with a loss of native 3d structure.
however, a few other cysteine cathepsins of the papain superfamily clan c1a display prolonged stability and activity at neutral ph values, cathepsins k and s are the most prominent examples  <cit> .
in contrast, papain as well as cruzain exhibit stability at broad ranges of ph values and withstand neutral to even basic ph-conditions  <cit> .
on the other hand, other plant proteases are particularly prone to turn instable at ph values differing from their acidic optimum  <cit> .
these pronounced differences in the ph-dependent stability indicate that papain-like enzymes have evolved  <cit>  to play roles in a variety of biological processes, bringing proteolysis into milieus outside acidic endo-lysosomal compartments.
therefore, mammalian cysteine cathepsins might be the "specialists" in acidic environments, although they are still suited to perform their actions, at least temporarily, under the neutral ph-conditions of the extracellular space that are nowadays considered non-favorable or even "extreme"  <cit> .
in fact, proteolytic activity of lysosomal cysteine peptidases in an environment exhibiting conditions opposite to the reducing and acidic milieu of endo-lysosomal compartments was not long ago considered to be a mission impossible.
oxidizing conditions enforce the formation of disulfide bonds by reactive thiol groups which makes the active site cysteine of cathepsins highly sensitive to oxidation.
thus, under reducing conditions, like it is assumed for the endo-lysosomal compartment, the activity of cysteine cathepsins should be considerably higher than in other compartments exhibiting oxidizing conditions, like e.g.
the extracellular space.
in the thyroid, however, the extracellular follicle lumen is the scene of action for the initial steps of thyroid hormone utilization, i.e.
the solubilization of covalently cross-linked tg and thyroxine liberation by proteolytic processing mediated primarily through cysteine cathepsins b, k, l and s  <cit> .
up to now, the hypothesis that cysteine peptidases hold proteolytic activity in oxidizing environments was rarely challenged experimentally due to the lack of suitable activity assays with conditions mimicking the in vivo situation in the most life-like fashion, i.e.
at realistic ph and redox conditions in physiological settings.
in situ, redox conditions in cellular compartments or in the extracellular space are achieved and maintained by redox pairs of which cysteine-cystine constitutes the prominent and natural redox-pair of the extracellular space and endo-lysosomal compartments  <cit> .
in keeping with this notion, exact values describing redox potentials are astonishingly scarce and have been reported for the cytosol  and the er lumen   <cit> .
the latter represents an oxidizing intracellular compartment of protein folding in which disulfide bridges are formed and maintained, whereas the cytosol is clearly reducing.
for the choice of our buffer system we used the - redox-pair to adjust the redox potential and we considered a redox potential of - <dig> mv suitable to simulate reducing conditions of endosomes and lysosomes.
a redox value of - <dig> mv was adjusted for mimicking the extracellular space, i.e.
we assumed a slightly more oxidizing milieu for the extracellular space than that observed for the er lumen.
through the use of a two-buffer system with citric acid and sodium phosphate and through the addition of l-cysteine, we have established buffers with stable redox potentials at distinct ph values for in vitro analyses of proteolytic substrate processing potency.
another critical factor in proteolytic profiling is the choice of substrate.
commonly, small synthetic substrates are used for standard protease activity assays.
more recently it became mandatory to use natural substrates in in vitro assays to collect more appropriate data; especially considering that protease-substrate interaction may also affect enzyme activity  <cit> .
the cleavage of tg by cathepsins is a naturally occurring process of high physiological importance  <cit> , which makes tg an excellent substrate in our proteolytic assays.
the partial degradation of extracellularly stored full-length tg within the thyroid follicle lumen is of great importance in vivo.
it represents the necessary first step to produce smaller fragments of the pro-hormone that can easily be endocytosed to ensure endo-lysosomal processing and efficient thyroid hormone liberation.
in the in vitro assay presented herein, partial proteolytic processing of full length tg was achieved by all cathepsins, resulting in the generation of three distinct fragments even at neutral and oxidizing conditions.
the abundance of low molecular mass tg-fragments in lysosomal-mimicking conditions of this assay must be considered representative of an extensive overall cleavage of the substrate.
it can therefore be deduced that all cysteine cathepsins tested exhibited highest proteolytic activities, indeed, under acidic and reducing conditions.
this notion is in line with the catabolic function of cysteine cathepsins in situ which is mainly conducted within the endo-lysosomal compartment.
we consider cathepsin l as the most potent peptidase to catabolize tg within the lysosomes, because the respective samples lacked full-length, intact tg almost entirely, but contained numerous smaller fragments of 40- <dig> kda.
the application of several cathepsins at once proved to be efficient in well-directed substrate cleavage further strengthening results from our previous in vivo studies in which it was shown that a concerted action of cathepsins k and l is needed for t <dig> liberation in vivo  <cit> .
the results presented herein further support the conclusion that tg-processing and thyroid hormone liberation is achieved by proteases acting consecutively towards efficient pro-hormone utilization.
cathepsin s proved to be the best-suited cysteine cathepsin to extracellularly liberate t <dig> from tg, i.e.
to conduct hormone liberation before the pro-hormone eventually reached endosomes and lysosomes.
cathepsin s was also able to liberate t <dig> under endosomal and lysosomal conditions, but then, it was not any more efficient than the other cysteine cathepsins tested.
in fact, it has been shown that cathepsin s is the only cysteine cathepsin which is truly stable at neutral ph when analyzed under reducing conditions, whereas cathepsins k and l are unstable and cathepsin b exhibits limited stability at reducing and neutral ph conditions  <cit> .
interestingly, an additional cleavage site for the combination of cathepsins k and s was determined within the n-terminal half of tg at extracellular conditions as compared to cleavage by cathepsin s alone .
this might explain why liberation of t <dig> under these conditions was less effective when both, cathepsins k and s were used in contrast to combinations of cathepsin s with cathepsins b or l, in which the cathepsin s ability to utilize t <dig> from tg clearly dominated .
however, it should be kept in mind that the assay conditions as presented herein differ significantly from those of previous studies in that we mimicked physiological conditions by testing cysteine cathepsins under oxidizing and neutral ph conditions.
therefore, our results gained by studying cleavage of their natural substrate tg under such comparatively "extreme" non-favorable conditions clearly differ from the standard assay conditions in which the read-out is not physiological, but instead cleavage of synthetic substrates is monitored.
hence, we believe that the data presented here has to be interpreted on the background of physiological protein processing with the biologically significant read-out, that is, utilization of t <dig> by processing of the precursor tg.
none of the cleavage sites used by any of the cysteine cathepsins tested was identical in both, extracellular and endosomal conditions, i.e.
in the compartments of well-directed substrate processing.
thus, we conclude that preferential cleavage is largely up to the compartment in which substrate processing takes place.
the presence of different sets of proteolytic enzymes in such compartments may allow for a combinatorial and sequential substrate cleavage.
this is to say that specific cleavage sites are used in distinct environments, which provide stable, biochemically defined conditions for specific sets of enzymes, but do not necessarily exhibit the most favorable conditions for bulk proteolytic processing.
this notion is further supported by comparative analysis of endosomal-typical cleavage with respect of lysosomal-typical cleavage sites, which completely lack analogies as has been determined by us  and by dunn and co-workers using isolated cathepsins or extracts of thyroid lysosomes  <cit> .
even though the cysteine cathepsins b and l were included in both studies, only one cleavage-site  was found to be alike in extracellular conditions  and in tg fragments identified in lysosomal extracts .
interestingly, this particular cleavage site was not reproduced by identical cathepsins in different environments, but the extracellular-representative cleavage of tg by cathepsin s  generated an almost identical neo-n-terminus as the cathepsin b-mediated cleavage determined by dunn and co-workers  <cit> .
thus, although the amino acid sequence of the substrate itself predominantly dictates its cleavage, the specificity of substrate cleavage is additionally directed by the biochemical environment of cleavage and by the set of actually cleaving enzymes.
it is important to keep in mind that the extracellularly acting cysteine cathepsins in the thyroid follicle are derived from their secretion into the follicle lumen after being recruited out of endo-lysosomal compartments  <cit> .
it is generally accepted that the proteolytic activity of such already activated cysteine peptidases can be maintained under oxidizing conditions in the extracellular space for longer time intervals  <cit> .
since the proteolytic enzymes cleaving tg in the thyroid follicle lumen have experienced a reducing and acidic environment for proper processing and activation before being secreted, they may as well contribute significantly to physiological thyroid hormone liberation by extracellular proteolysis of tg.
CONCLUSIONS
our study shows that a specific subcellular location is the key figure in the nature of proteolytic processing, i.e.
timing and location of peptidase activities determine whether bulk protein turnover or, instead, specific substrate processing takes place.
importantly enough, the onset of many pathological processes like tumor cell metastasis, rheumatoid arthritis, pancreatitis or multiple sclerosis goes along with alterations in the patterns of cysteine cathepsin localization and with pericellular acidification which may well be suited to prolong the extracellular actions of these proteases .
the routine to analyze cysteine cathepsin-mediated processing of tg presented herein will be easily adaptable to investigate any protease's proteolytic potential towards any natural or artificial substrate.
hence, our approach is applicable in order to study the degradome in a physiological context at simulated in vivo conditions.
by adapting our assay system to other proteases, we anticipate a better understanding of the interplay and fine-adjustments of protease-networks that control substrate processing in the determination of 'vital' or 'fatal' changes in cellular destiny.
comprehensive knowledge of proteolysis in physiological or pathological conditions, in turn, is critical for the development of effective treatments and novel therapeutics.

 proteolytic processes are of vital importance due to the irreversibility of peptide bond cleavage. spatio-temporal regulation of proteolysis is therefore of highest significance in determining the fate of cells. to date more than gene sequences have been annotated as coding for proteolytic enzymes. the diversity of proteases is further reflected in broad tissue-, compartment- and substrate specificities. most of all protease genes encode for serine peptidases, followed by metallo and cysteine peptidases. because of this variety, one key issue of degradomics is to investigate networks of proteases in order to explain distinct proteolytic functions in the spatio-temporal context of substrate cleavage. it is common practice to employ proteolytic activity assays to achieve this goal. in this report we focused on cysteine cathepsins b, k, l and s, and analyzed their processing patterns using a natural substrate, thyroglobulin, while taking into account the suspected redox- and ph values of the physiological cleavage environments. it has been shown before that the cysteine cathepsins b, k, l and s are localized to the endo-lysosomal system of thyroid epithelial cells. moreover, they are present at extracellular locations such as the apical plasma membrane and within the follicular lumen of the thyroid gland. generally, cysteine cathepsins of the papain superfamily are well known to engage in bulk protein turnover at the increasingly acidic ph values of endocytic compartments. nevertheless, the proteolytic potency of cysteine cathepsins towards peptidic or natural substrates has been demonstrated to expand over a wide range including neutral ph values. in fact, cysteine cathepsins were often accredited as extracellularly acting enzymes in vivo. diverse protocols are available that describe methods to perform activity assays of cysteine cathepsins at neutral conditions, usually after activation of the proteases by incubation with reducing agents. in reality, however, the extracellular space is not only a neutral but also an oxidizing environment, as much as endosomes and lysosomes exhibit not only acidic but also reducing conditions. in this study, we have chosen redox-values of - mv to mimic the reducing environment of the cysteine-rich endosomes and lysosomes. moreover, endo-lysosomes are characterized by increasingly acidic conditions, and we considered ph and ph as closely reflecting the endosomal and lysosomal milieu, respectively. on the other hand, the thyroid follicle lumen represents a secluded compartment in which the tight junctions between thyroid epithelial cells tie up the extracellular storage space for tg from the intercellular spaces between thyrocytes. the follicle lumen is assumed to be neutral in ph thereby rendering tg proteolysis by extracellular hydrolases unlikely. however, more recently we provided evidence for tg proteolysis mediated by cysteine cathepsins within the extracellular thyroid follicle lumen before its endocytosis. extracellularly stored tg of the human thyroid follicle lumen is covalently cross-linked by disulfide bonds which can be formed in a self-assisted fashion via the cxxc-boxes within tg, thereby attesting an oxidizing milieu within the thyroid follicle lumen. furthermore, the direct pericellular environment adjacent to the apical plasma membrane of thyroid epithelial cells is oxidizing due to the presence of an h2o generating system which enables thyroid peroxidase to iodinate tg at neutral ph. we have chosen a value of - mv to mimic the extracellular space which is slightly more oxidizing than the range of - mv to - mv that was reported for the lumen of the endoplasmic reticulum. as yet, there are only very few studies which consider both, ph values and redox potentials of the cleavage environment. the assay described herein therefore employs buffers accounting for the conditions in the follicle lumen equivalent to the extracellular space or within endo-lysosomal compartments. we assessed the suitability of such buffers to promote proteolytic cleavage of tg by specific cysteine cathepsins in order to simulate proteolysis within thyroid follicles, i.e. within an extracellular compartment, or within the endo-lysosomes of thyrocytes. processing of tg in the thyroid serves as an excellent model system for our purposes, because it has been demonstrated that the cysteine cathepsins b, k, l and s cleave tg in vitro, tg is located in all compartments where cysteine cathepsins principally meet their substrates, i.e. endosomes, lysosomes, and in the extracellular space, cysteine cathepsins and tg represent a naturally occurring and physiologically relevant enzyme-substrate couple in vivo. under physiological conditions, proteolysis of tg leading to thyroid hormone liberation is accomplished by cysteine cathepsins in a sequential process starting with the solubilization of tg from its covalently cross-linked macromolecular storage forms that fill the extracellular follicle lumen. the initial substrate cleavage results in the production of smaller protein fragments that are internalized by thyroid epithelial cells together with partially processed tg. accordingly, liberation of the thyroid hormones t and t which are essential for the regulation of mammalian development and metabolism begins extracellularly and is perpetuated within endocytic compartments of thyroid epithelial cells. this study aims at elucidating crucial roles of selected cysteine cathepsins in this sequence of prohormone processing. the development of an in vitro assay system mimicking the in situ ph and redox conditions enabled analysis of the potency of cathepsins b, k, l and s to cleave their natural substrate tg and to liberate thyroid hormones under conditions simulating favorable versus non-favorable compartments.  localization of cysteine cathepsins in the human thyroid gland the subcellular localization of cysteine cathepsins has been studied in detail in almost all tissues but not to the same extent in the human thyroid gland. therefore and in order to analyze the distribution patterns of particular cysteine cathepsins in the compartments of tg processing, human thyroid tissue was immunolabeled with antibodies against cathepsins b, k, l, and s in single and double labeling experiments. cathepsins b, k, and l were co-localized within the same intracellular vesicles. cathepsin s appeared to be also localized to distinct vesicular compartments, because co-localization with cathepsins k and l was not as prominent. some cathepsin l-positive vesicles were even lacking cathepsin s. likewise, cathepsin k-positive structures were often negative for cathepsin s. consequently, cathepsin s-positive vesicles lacking cathepsins k or l were frequently detected. moreover, all cathepsins analyzed were detected in the extracellular space, i.e. within the follicle lumen, indicating their secretion. note, that the extracellular follicle lumen represents a storage compartment for tg, hence, all cathepsins are found in close proximity to their natural substrate already here. cathepsin s was prominently present in the extracellular follicle lumen. cathepsins k and l were additionally found pericellularly in close association with the apical plasma membrane, where thyroid hormone liberation by tg processing is believed to start. we conclude that trafficking of proteases in thyroid epithelial cells results in a distinct protease composition within vesicles of the endocytic pathway in addition to their secretion into the extracellular space. hence, it is important to analyze tg processing with different combinations of cysteine cathepsins and to simulate substrate cleavage at the conditions of the respective compartments, i.e. extracellular follicle space, endosomes and lysosomes. establishment of buffers with stable redox potentials at distinct ph values buffers representative for the extracellular space, the endosomal compartment and for lysosomes were established through the use of a two-buffer system of citric acid, sodium phosphate, and l-cysteine. in order to verify buffer stability, the actual ph and redox values were determined for a minimum time period of hours. the buffer simulating extracellular conditions with an oxidizing redox potential of - mv and neutral ph was shown to be stable over hours. although addition of tg marginally lowered redox and ph values, the overall stability under extracellular conditions was more stringent in the presence of substrate. buffers mimicking endosomes and lysosomes were adjusted to - mv and differed from one another in their ph values, only. the endosomal-representative buffer was set to ph and the lysosomal to ph. the stability of these buffers with respect to redox potential and ph value was verified over a h time period as these buffers were stable from the very beginning. determination of cleavage sites within tg isolated tg was incubated with cathepsins in buffers simulating extracellular conditions and the extent of substrate cleavage was compared to that achieved under conditions characteristic for intracellular proteolysis within endo-lysosomal compartments. cathepsins b, k, l and s or combinations thereof were incubated with tg in the respective buffers and proteolysis was allowed to occur for hours. preparations were then heated in lds sample buffer without any further addition of reducing agents. this procedure ensured consistent behavior of all samples from the same conditions upon separation by page since they were run on separate gels. fragmentation patterns were analyzed by sds-page followed by immunoblotting using antibodies specific for tg and by n-terminal sequencing of selected tg-fragments. note, that for all incubations an identical amount of tg was subjected to cysteine cathepsin degradation, whereby the particular amount of distinct enzymes was kept constant in all incubations. therefore, the ratios of total enzyme to substrate differed between single and combinatorial incubations. we were especially decided about such conditions, because we consider this setup to best reflect tg cleavage in situ. as we have shown in the localization studies, vesicles may contain only one type of enzyme or combinations of different enzymes. for the extracellular follicle lumen, similar approximations might be relevant, since we observed almost no co-localization of the distinct cysteine cathepsins in the extracellular space, but different enzymes were detectable and can therefore, in principle, cleave luminal tg. hence, the observed degradation patterns must be interpreted as resulting from different combinations of enzymes acting sequentially and/or at the same time on tg. under neutral and oxidizing conditions mimicking the extracellular thyroid follicle lumen full-length tg of approximately kda was cleaved by each of the cysteine cathepsins tested, even though these buffer conditions are considered as'non-favorable' for proteolytic action of cysteine cathepsins. the effectiveness of substrate-cleavage was obvious from the appearance of two fragments of approximately and kda, which were also detected when the assay buffers reflected more favorable endosomal cleavage conditions, i.e. a reducing and slightly acidic environment. in addition to the high molecular mass fragments, all samples containing cathepsin s displayed a specific kda fragment under extracellular conditions. the same-sized fragment was generated at conditions characteristic for endosomes and lysosomes by several combinations of cysteine cathepsins but notably not by cathepsin k alone or in combination with cathepsins l and s. the analysis of degradation patterns using assay conditions mimicking lysosomal cleavage revealed a number of low molecular mass fragments of tg as generated by all cathepsin combinations. samples containing cathepsin l lacked the intact, full-length tg, but contained numerous smaller fragments of 40- kda. note that all preparations contained equal amounts of tg at the start of incubation with cysteine cathepsins under different conditions. the lanes were loaded after the incubation period with identically sized aliquots from each preparation. hence, the absence of signal in certain lanes of the immunoblots is representative of extensive tg degradation rather than being the result of unequal loading of the gels. accordingly, blank or weakly labeled lanes indicate complete degradation of tg by the respective cysteine cathepsin/s at these conditions. as a control of its stability, tg was incubated in the respective buffers, but without any protease added. in order to determine the cleavage sites that were used by cysteine cathepsins, characteristic tg fragments were n-terminally sequenced. a band with a molecular mass of approximately kda exhibited the same n-terminus as unprocessed tg in all instances, thus representing intact, full-length protein. tg-fragments originating from proteolytic processing by cathepsin s under extracellular conditions had molecular masses of approximately kda, kda and kda. two of these fragments exhibited newly generated n-termini, while the kda fragment showed the same n-terminus as unprocessed tg. arrowheads indicate relative positions of identified cleavage sites as deduced from n-terminal sequencing of extracellular- or endosomal-like tg degradation. in figure 5d a scheme of lysosomal cleavage sites is depicted, which was determined by dunn and colleagues under near-physiological conditions since lysosomal extracts were used to degrade tg. the schematic drawings in figure illustrate that the cleavage sites varied within tg depending on the conditions under which cleavage took place. analysis of physiological significance of tg cleavage determination of cathepsin cleavage sites revealed that the n-terminal half of tg was prone to extracellular and endosomal cleavage, whereas the thyroid hormone-rich c-terminal half is, according to, best cleaved under conditions of lysosomal tg processing. in order to implement a physiologically relevant read-out for efficiency of substrate cleavage, we analyzed the in vitro-degradation samples for their content of liberated, free thyroxine by elisa. under extracellular conditions, ft was only detected in samples that contained cathepsin s alone or in combinations with other cathepsins. under endosomal conditions, the overall efficiency of ft liberation was considerable, about fold higher than compared to extracellular conditions with values of ft ranging from approximately to pm. yet, another augmentation in the amount of liberated t was observed under conditions simulating lysosomes where cathepsins b, k and l performed equally well by liberating t in a range from to pm. discussion one important mechanism to control protease activity is given by endogenous inhibitors, another is represented by the dependency of proteolytic enzymes on the ph and redox potential of the cleavage environment. whereas ph sensitivity has been studied quite extensively, much less is known about their sensitivity to the redox potential of the environment. endo-lysosomal cysteine proteases like cathepsins l and b are considered highly instable at neutral ph values with half lives of and minutes, respectively. the inactivation appears to be irreversible and is associated with a loss of native 3d structure. however, a few other cysteine cathepsins of the papain superfamily clan c1a display prolonged stability and activity at neutral ph values, cathepsins k and s are the most prominent examples. in contrast, papain as well as cruzain exhibit stability at broad ranges of ph values and withstand neutral to even basic ph-conditions. on the other hand, other plant proteases are particularly prone to turn instable at ph values differing from their acidic optimum. these pronounced differences in the ph-dependent stability indicate that papain-like enzymes have evolved to play roles in a variety of biological processes, bringing proteolysis into milieus outside acidic endo-lysosomal compartments. therefore, mammalian cysteine cathepsins might be the "specialists" in acidic environments, although they are still suited to perform their actions, at least temporarily, under the neutral ph-conditions of the extracellular space that are nowadays considered non-favorable or even "extreme". in fact, proteolytic activity of lysosomal cysteine peptidases in an environment exhibiting conditions opposite to the reducing and acidic milieu of endo-lysosomal compartments was not long ago considered to be a mission impossible. oxidizing conditions enforce the formation of disulfide bonds by reactive thiol groups which makes the active site cysteine of cathepsins highly sensitive to oxidation. thus, under reducing conditions, like it is assumed for the endo-lysosomal compartment, the activity of cysteine cathepsins should be considerably higher than in other compartments exhibiting oxidizing conditions, like e.g. the extracellular space. in the thyroid, however, the extracellular follicle lumen is the scene of action for the initial steps of thyroid hormone utilization, i.e. the solubilization of covalently cross-linked tg and thyroxine liberation by proteolytic processing mediated primarily through cysteine cathepsins b, k, l and s. up to now, the hypothesis that cysteine peptidases hold proteolytic activity in oxidizing environments was rarely challenged experimentally due to the lack of suitable activity assays with conditions mimicking the in vivo situation in the most life-like fashion, i.e. at realistic ph and redox conditions in physiological settings. in situ, redox conditions in cellular compartments or in the extracellular space are achieved and maintained by redox pairs of which cysteine-cystine constitutes the prominent and natural redox-pair of the extracellular space and endo-lysosomal compartments. in keeping with this notion, exact values describing redox potentials are astonishingly scarce and have been reported for the cytosol and the er lumen. the latter represents an oxidizing intracellular compartment of protein folding in which disulfide bridges are formed and maintained, whereas the cytosol is clearly reducing. for the choice of our buffer system we used the - redox-pair to adjust the redox potential and we considered a redox potential of - mv suitable to simulate reducing conditions of endosomes and lysosomes. a redox value of - mv was adjusted for mimicking the extracellular space, i.e. we assumed a slightly more oxidizing milieu for the extracellular space than that observed for the er lumen. through the use of a two-buffer system with citric acid and sodium phosphate and through the addition of l-cysteine, we have established buffers with stable redox potentials at distinct ph values for in vitro analyses of proteolytic substrate processing potency. another critical factor in proteolytic profiling is the choice of substrate. commonly, small synthetic substrates are used for standard protease activity assays. more recently it became mandatory to use natural substrates in in vitro assays to collect more appropriate data; especially considering that protease-substrate interaction may also affect enzyme activity. the cleavage of tg by cathepsins is a naturally occurring process of high physiological importance, which makes tg an excellent substrate in our proteolytic assays. the partial degradation of extracellularly stored full-length tg within the thyroid follicle lumen is of great importance in vivo. it represents the necessary first step to produce smaller fragments of the pro-hormone that can easily be endocytosed to ensure endo-lysosomal processing and efficient thyroid hormone liberation. in the in vitro assay presented herein, partial proteolytic processing of full length tg was achieved by all cathepsins, resulting in the generation of three distinct fragments even at neutral and oxidizing conditions. the abundance of low molecular mass tg-fragments in lysosomal-mimicking conditions of this assay must be considered representative of an extensive overall cleavage of the substrate. it can therefore be deduced that all cysteine cathepsins tested exhibited highest proteolytic activities, indeed, under acidic and reducing conditions. this notion is in line with the catabolic function of cysteine cathepsins in situ which is mainly conducted within the endo-lysosomal compartment. we consider cathepsin l as the most potent peptidase to catabolize tg within the lysosomes, because the respective samples lacked full-length, intact tg almost entirely, but contained numerous smaller fragments of 40- kda. the application of several cathepsins at once proved to be efficient in well-directed substrate cleavage further strengthening results from our previous in vivo studies in which it was shown that a concerted action of cathepsins k and l is needed for t liberation in vivo. the results presented herein further support the conclusion that tg-processing and thyroid hormone liberation is achieved by proteases acting consecutively towards efficient pro-hormone utilization. cathepsin s proved to be the best-suited cysteine cathepsin to extracellularly liberate t from tg, i.e. to conduct hormone liberation before the pro-hormone eventually reached endosomes and lysosomes. cathepsin s was also able to liberate t under endosomal and lysosomal conditions, but then, it was not any more efficient than the other cysteine cathepsins tested. in fact, it has been shown that cathepsin s is the only cysteine cathepsin which is truly stable at neutral ph when analyzed under reducing conditions, whereas cathepsins k and l are unstable and cathepsin b exhibits limited stability at reducing and neutral ph conditions. interestingly, an additional cleavage site for the combination of cathepsins k and s was determined within the n-terminal half of tg at extracellular conditions as compared to cleavage by cathepsin s alone. this might explain why liberation of t under these conditions was less effective when both, cathepsins k and s were used in contrast to combinations of cathepsin s with cathepsins b or l, in which the cathepsin s ability to utilize t from tg clearly dominated. however, it should be kept in mind that the assay conditions as presented herein differ significantly from those of previous studies in that we mimicked physiological conditions by testing cysteine cathepsins under oxidizing and neutral ph conditions. therefore, our results gained by studying cleavage of their natural substrate tg under such comparatively "extreme" non-favorable conditions clearly differ from the standard assay conditions in which the read-out is not physiological, but instead cleavage of synthetic substrates is monitored. hence, we believe that the data presented here has to be interpreted on the background of physiological protein processing with the biologically significant read-out, that is, utilization of t by processing of the precursor tg. none of the cleavage sites used by any of the cysteine cathepsins tested was identical in both, extracellular and endosomal conditions, i.e. in the compartments of well-directed substrate processing. thus, we conclude that preferential cleavage is largely up to the compartment in which substrate processing takes place. the presence of different sets of proteolytic enzymes in such compartments may allow for a combinatorial and sequential substrate cleavage. this is to say that specific cleavage sites are used in distinct environments, which provide stable, biochemically defined conditions for specific sets of enzymes, but do not necessarily exhibit the most favorable conditions for bulk proteolytic processing. this notion is further supported by comparative analysis of endosomal-typical cleavage with respect of lysosomal-typical cleavage sites, which completely lack analogies as has been determined by us and by dunn and co-workers using isolated cathepsins or extracts of thyroid lysosomes. even though the cysteine cathepsins b and l were included in both studies, only one cleavage-site was found to be alike in extracellular conditions and in tg fragments identified in lysosomal extracts. interestingly, this particular cleavage site was not reproduced by identical cathepsins in different environments, but the extracellular-representative cleavage of tg by cathepsin s generated an almost identical neo-n-terminus as the cathepsin b-mediated cleavage determined by dunn and co-workers. thus, although the amino acid sequence of the substrate itself predominantly dictates its cleavage, the specificity of substrate cleavage is additionally directed by the biochemical environment of cleavage and by the set of actually cleaving enzymes. it is important to keep in mind that the extracellularly acting cysteine cathepsins in the thyroid follicle are derived from their secretion into the follicle lumen after being recruited out of endo-lysosomal compartments. it is generally accepted that the proteolytic activity of such already activated cysteine peptidases can be maintained under oxidizing conditions in the extracellular space for longer time intervals. since the proteolytic enzymes cleaving tg in the thyroid follicle lumen have experienced a reducing and acidic environment for proper processing and activation before being secreted, they may as well contribute significantly to physiological thyroid hormone liberation by extracellular proteolysis of tg.  our study shows that a specific subcellular location is the key figure in the nature of proteolytic processing, i.e. timing and location of peptidase activities determine whether bulk protein turnover or, instead, specific substrate processing takes place. importantly enough, the onset of many pathological processes like tumor cell metastasis, rheumatoid arthritis, pancreatitis or multiple sclerosis goes along with alterations in the patterns of cysteine cathepsin localization and with pericellular acidification which may well be suited to prolong the extracellular actions of these proteases. the routine to analyze cysteine cathepsin-mediated processing of tg presented herein will be easily adaptable to investigate any protease's proteolytic potential towards any natural or artificial substrate. hence, our approach is applicable in order to study the degradome in a physiological context at simulated in vivo conditions. by adapting our assay system to other proteases, we anticipate a better understanding of the interplay and fine-adjustments of protease-networks that control substrate processing in the determination of'vital' or'fatal' changes in cellular destiny. comprehensive knowledge of proteolysis in physiological or pathological conditions, in turn, is critical for the development of effective treatments and novel therapeutics. 
therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and ph values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. the proteolytic potencies of cysteine cathepsins b, k, l and s towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic ph values. cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. the results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. in particular, proteolytic activity of cathepsin s towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. we conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays. moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin s at extracellular conditions, while cathepsins b, k and l worked most efficiently in this respect at endo-lysosomal conditions. thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. all analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by sds-page followed by immunoblotting or n-terminal sequencing.
 cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and ph values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells.  the proteolytic potencies of cysteine cathepsins b, k, l and s towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic ph values. thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. all analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by sds-page followed by immunoblotting or n-terminal sequencing. moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin s at extracellular conditions, while cathepsins b, k and l worked most efficiently in this respect at endo-lysosomal conditions.  the results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. in particular, proteolytic activity of cathepsin s towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. we conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays. 