No description available.

LD2668 .T4 1948 D5 / Master of Science

A proteolytic activity has been shown to be associated with the heat shock protein 70 (hsp70). In order to study this, I have constructed RNA transcribing vectors with the coding sequences of the D. melanogaster (pBUG7) and the human (pMAN70) genes coding hsp70, and with an internal deletion (pBUG301) in D. melanogaster. Proteins from 37 kDa to 70 kDa were translated in a rabbit reticulocyte lysate in the presence of 35S-methionine from RNA synthesized in vitro off the full length templates (pBUG7, and pMAN70), or altered templates. Restriction digestion of pBUG7 with BamH I and Nar I yields templates that produce carboxy-terminal truncated proteins of 37 kDa and 61 kDa respectively. The full length and the truncated proteins contain a proteolytic activity when assayed by SDS/PAGE in two dimensions. The internally deleted protein does not maintain the proteolytic activity. The proteolytic activity was shown not to be the result of non-enzymatic cleavage. A general serine proteinase inhibitor eliminates the proteolytic activity of the full length human and D. melanogaster hsp70. This evidence shows that the proteolytic activity is directly connected to hsp70.

No description available.

Proteolytic processes are often mediated by highly orchestrated cascades, through which protease enzymes function coordinately to ensure a stepwise activation. This thesis presents experimental data which supports and complements the previously postulated mechanism of KLK (kallikrein-related protease) activation through proteolytic cascades. Further examination of the seminal KLK cascade has revealed several of its key (patho) physiological roles in human reproductive system. Multiple members of the seminal KLK cascade, in particular KLK14, were shown to play a pivotal role in regulating semen liquefaction. The cascade was further shown to be tightly regulated through a series of highly orchestrated feedback loops, to prevent deleterious effects due to aberrant protease activation. Accordingly, a strong association was observed between the expression level of several seminal KLKs, delayed liquefaction, and other markers of semen quality, including semen hyperviscosity. Furthermore, a strong association was found between delayed liquefaction and abnormal sperm motility. Therefore, dysregulated KLK expressions and/or activities were proposed as an underlying cause of male subfertility. Finally, this thesis has provided initial insights into a novel potential function of multiple members of the seminal KLK cascade in activation of the key immune-deviating agent, TGFβ1, in seminal plasma. TGFβ1 activation is postulated to be mediated directly through complete fragmentation or indirectly through partial cleavage and conformational changes of the LAP propeptide motif of the latent TGFβ1. KLK- mediate proteolytic cleavage of the TGFβ1 binding protein, LTBP1, is also suggested as a potential physiological mechanism for release of the membrane-bound latent TGFβ1. Overall, the data provided here may suggest a common regulatory mechanism, involved co-temporally in the two key processes of semen liquefaction and immune-suppression. This might be critical in protecting motile sperms following their release from semen coagulum. Understanding KLK-mediated proteolytic events in seminal plasma can shed light not only on the physiological role of this family of enzymes, but also on some of causes of male subfertility. Accordingly, therapeutic induction of this cascade may be utilized to supplement the current clinical treatment of male subfertility. Conversely, targeted inhibition of key components of the cascade may have potential pharmaceutical utility as a novel topical contraceptive strategy.

The main site for protein degradation along the endosomal pathway is believed to be the late endosome. Lysosomes are thought to be storage organelles that, when necessary, inject proteases into the late endosome. It was hypothesised that differences in the lumenal redox environments between the two organelles could be responsible for their functional differences. In an attempt to quantify this potential difference, the lysosomal cysteine protease cathepsin B was isolated by an improved purification procedure. Several intracellular reducing agents were used to activate cathepsin B, the most effective being cysteine. Cysteine was used to activate cathepsin B under various pH conditions in order to model endosomal conditions. An inverse relationship was found between the pH and the concentration of cysteine required to activate cathepsin B. This suggested that cathepsin B may have an optimal redox potential. In order to determine this potential, cysteinexystine redox buffers were made up and used in determination of the activity of the enzyme against a synthetic and a whole protein substrate (haemoglobin). No distinct redox potential could be determined using either substrate, but it was found that cystine stimulated proteolysis of haemoglobin. A similar stimulatory effect was observed for cathepsin D and papain hydrolysis of haemoglobin. This effect is possibly due to the ability of cystine to promote substrate structure, effectively increasing the substrate concentration. These findings and other results obtained from the literature have been used to create a model of how proteolysis may be regulated along the endosomal system. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. August 2014. / Human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), remains a topic of global concern even though great strides have been made to combat the virus. The high replicative rate of the virus and recombination of the variety of viral strains complicate the treatment of AIDS. There has been an increasing prevalence of African HIV strains in the Americas and Europe. The viral protease (PR) is vital for the propagation of the virus; and thus, is a major target in antiviral therapy. The HIV-1 PR enzyme from the subtype C strain; which predominates in sub- Saharan Africa, has been greatly under-investigated in comparison to the protease from the subtype B strain which predominates in North America and Europe. Enzyme activity data which were part of this work suggested that the South African HIV-1 subtype C protease (CSA PR) displays improved substrate turnover in comparison to the subtype B PR. Thermodynamics and inhibition kinetics of drug binding showed that the C-SA PR is less susceptible to certain clinically-used protease inhibitors when compared to the subtype B PR. A crystal structure of the C-SA PR was solved and showed no difference to the global structure of the subtype B PR. Molecular dynamics simulations showed that the C-SA PR exhibits a wider range of open conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) was performed to elucidate the mechanism of reduced drug susceptibility displayed by the C-SA PR. HDX-MS data provided insights into the basis of the increased preference for open conformers displayed by the C-SA PR and the stability of the terminal dimer interface which is a target in protease inhibition.

Abstract not available

Abstract not available

Graduation date: 1996