The ability to tolerate multiple host derived stresses, resist eradication and persist within the infected individuals is central to the pathogenicity of Mycobacterium tuberculosis (Mtb), the ...causative agent of tuberculosis (TB). Mycobacterial survival is contingent upon sensing environmental perturbations and initiating a fitting response to counter them. Therefore, understanding of molecular mechanisms underlying stress tolerance and sensing in Mtb is critical for devising strategies for TB control. Our study aims to delineate the role of ClpB, a heat shock protein of Hsp100 family, in the general stress response and persistence mechanisms of Mtb. We demonstrate that Mtb requires ClpB to survive under stressful conditions. Additionally, we show that ClpB is necessary for the bacteria to persist in latency-like conditions such as prolonged hypoxia and nutrient-starvation. The disruption of ClpB results in aberrant cellular morphology, impaired biofilm formation and reduced infectivity of Mtb ex vivo. Our study also reports an alternative role of ClpB as a chaperokine which elicits inflammatory response in host. We conclude that ClpB is essential for Mtb to survive within macrophages, and plays a crucial part in the maintenance of dormant Mtb bacilli in latent state. The absence of ClpB in human genome makes it an attractive choice as drug target for TB.
Understanding the antibody response to SARS-CoV-2, the virus responsible for COVID-19, is crucial to comprehending disease progression and the significance of vaccine and therapeutic development. The ...emergence of highly contagious variants poses a significant challenge to humoral immunity, underscoring the necessity of grasping the intricacies of specific antibodies. This review emphasizes the pivotal role of antibodies in shaping immune responses and their implications for diagnosing, preventing, and treating SARS-CoV-2 infection. It delves into the kinetics and characteristics of the antibody response to SARS-CoV-2 and explores current antibody-based diagnostics, discussing their strengths, clinical utility, and limitations. Furthermore, we underscore the therapeutic potential of SARS-CoV-2-specific antibodies, discussing various antibody-based therapies such as monoclonal antibodies, polyclonal antibodies, anti-cytokines, convalescent plasma, and hyperimmunoglobulin-based therapies. Moreover, we offer insights into antibody responses to SARS-CoV-2 vaccines, emphasizing the significance of neutralizing antibodies in order to confer immunity to SARS-CoV-2, along with emerging variants of concern (VOCs) and circulating Omicron subvariants. We also highlight challenges in the field, such as the risks of antibody-dependent enhancement (ADE) for SARS-CoV-2 antibodies, and shed light on the challenges associated with the original antigenic sin (OAS) effect and long COVID. Overall, this review intends to provide valuable insights, which are crucial to advancing sensitive diagnostic tools, identifying efficient antibody-based therapeutics, and developing effective vaccines to combat the evolving threat of SARS-CoV-2 variants on a global scale.
Mycobacterium tuberculosis (Mtb) is known to persist in extremely hostile environments within host macrophages. The ability to withstand such proteotoxic stress comes from its highly conserved ...molecular chaperone machinery. ClpB, a unique member of the AAA+ family of chaperones, is responsible for resolving aggregates in Mtb and many other bacterial pathogens. Mtb produces two isoforms of ClpB, a full length and an N‐terminally truncated form (ClpB∆N), with the latter arising from an internal translation initiation site. It is not clear why this internal start site is conserved and what role the N‐terminal domain (NTD) of Mtb ClpB plays in its function. In the current study, we functionally characterized and compared the two isoforms of Mtb ClpB. We found the NTD to be dispensable for oligomerization, ATPase activity and prevention of aggregation activity of ClpB. Both ClpB and ClpB∆N were found to be capable of resolubilizing protein aggregates. However, the efficiency of ClpB∆N at resolubilizing higher order aggregates was significantly lower than that of ClpB. Further, ClpB∆N exhibited reduced affinity for substrates as compared to ClpB. We also demonstrated that the surface of the NTD of Mtb ClpB has a hydrophobic groove that contains four hydrophobic residues: L97, L101, F140 and V141. These residues act as initial contacts for the substrate and are crucial for stable interaction between ClpB and highly aggregated substrates.
The chaperone ClpB resolves protein aggregates in many bacterial pathogens including Mtb. ClpB exists in a repressed state, and binding of DnaKJE proteins and aggregated substrate to ClpB's core orients its N‐terminal domain (NTD) towards substrate leading to de‐repression. The NTD makes initial contact with the substrate in the resolubilization process of ClpB. Lack of an NTD leads to compromised resolubilization of large aggregates.
Cancer remains the leading cause of global mortality, prompting a paradigm shift in its treatment and outcomes with the advent of targeted therapies. Among the most prevalent mutations in RAS-driven ...cancers, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations account for approximately 86% of cases worldwide, particularly in lung, pancreatic, and colon cancers, contributing to poor prognosis and reduced overall survival. Despite numerous efforts to understand the biology of KRAS mutants and their pivotal role in cancer development, the lack of well-defined drug-binding pockets has deemed KRAS an "undruggable" therapeutic target, presenting significant challenges for researchers and clinicians alike. Through significant biochemical and technological advances, the last decade has witnessed promising breakthroughs in targeted therapies for KRAS-mutated lung, colon, and pancreatic cancers, marking a critical turning point in the field. In this chapter, we provide an overview of the characteristics of KRAS mutations across various solid tumors, highlighting ongoing cutting-edge research on the immune microenvironment, the development of KRAS-driven mice models, and the recent progress in the exploration of specific KRAS mutant-targeted therapeutic approaches. By comprehensive understanding of the intricacies of KRAS signaling in solid tumors and the latest therapeutic developments, this chapter will shed light on the potential for novel therapeutic strategies to combat KRAS-driven tumors and improve patient outcomes.
MicroRNAs (miRNAs) are increasingly recognized as central players in the regulation of eukaryotic physiological processes. These small double stranded RNA molecules have emerged as pivotal regulators ...in the intricate network of cellular signaling pathways, playing significant roles in the development and progression of human cancers. The central theme in miRNA-mediated regulation of signaling pathways involves their ability to target and modulate the expression of pathway components. Aberrant expression of miRNAs can either promote or suppress key signaling events, influencing critical cellular processes such as proliferation, apoptosis, angiogenesis, and metastasis. For example, oncogenic miRNAs often promote cancer progression by targeting tumor suppressors or negative regulators of signaling pathways, thereby enhancing pathway activity. Conversely, tumor-suppressive miRNAs frequently inhibit oncogenic signaling by targeting key components within these pathways. This complex regulatory crosstalk underscores the significance of miRNAs as central players in shaping the signaling landscape of cancer cells. Furthermore, the therapeutic implications of targeting miRNAs in cancer are substantial. miRNAs can be manipulated to restore normal signaling pathway activity, offering a potential avenue for precision medicine. The development of miRNA-based therapeutics, including synthetic miRNA mimics and miRNA inhibitors, has shown promise in preclinical and clinical studies. These strategies aim to either enhance the activity of tumor-suppressive miRNAs or inhibit the function of oncogenic miRNAs, thereby restoring balanced signaling and impeding cancer progression. In conclusion, the crosstalk between miRNAs and signaling pathways in human cancers is a dynamic and influential aspect of cancer biology. Understanding this interplay provides valuable insights into cancer development and progression. Harnessing the therapeutic potential of miRNAs as regulators of signaling pathways opens up exciting opportunities for the development of innovative cancer treatments with the potential to improve patient outcomes. In this chapter, we provide an overview of the crosstalk between miRNAs and signaling pathways in the context of cancer and highlight the potential therapeutic implications of targeting this regulatory interplay.
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis when infects the host encounters several stresses within the host, resulting in aggregation of its proteins. To resolve this ...problem Mtb uses chaperones to either repair the damage or degrade the aggregated proteins. Mtb caseinolytic protein B (ClpB) helps in the prevention of aggregation and also resolubilization of aggregated proteins in bacteria, which is important for the survival of Mtb in the host. To function optimally, ClpB associates with its co-partners DnaK, DnaJ, and GrpE. The role of N-terminal domain (NTD) of Mtb ClpB in its function is not well understood. In this context, we investigated the interaction of three substrate mimicking peptides with the NTD of Mtb ClpB in silico. A substrate binding pocket, within the NTD of ClpB comprising of residues L136, R137, E138, K142, R144, R148, V149, Y158, and Y162 forming an ɑ-helix was thus identified. The residues L136 and R137 of the ɑ-helix were found to be important for the interaction of DnaK to ClpB. Further, nine single alanine recombinant variants of the identified residues were generated. As compared to the wild-type Mtb ClpB all the Mtb ClpB variants generated in this study were found to have reduced ATPase and protein refolding activity indicating the importance of the substrate binding pocket in ClpB function. The study demonstrates that the NTD of Mtb ClpB is important for its substrate interaction activity, and the substrate binding pocket identified in this study plays a crucial role in this interaction.
Communicated by Ramaswamy H. Sarma
Molecular chaperones are a conserved family of proteins that are over-expressed in response to heat and other stresses. The regulation of expression of chaperone proteins plays a vital role in ...pathogenesis of various bacterial pathogens. In M. tuberculosis, HrcA and HspR negatively regulate heat shock protein operons by binding to their cognate DNA elements, CIRCE and HAIR respectively. In this study, we show that M. tuberculosis HrcA is able to bind to its cognate CIRCE DNA element present in the upstream regions of groES and groEL2 operons only with the help of other protein(s). It is also demonstrated that M. tuberculosis HrcA binds to a CIRCE like DNA element present in the upstream region of hrcA gene suggesting its auto-regulatory nature. In addition, we report the presence of a putative HAIR element in the upstream region of groES operon and demonstrate the binding of HspR to it. In vitro, HrcA inhibited the DNA binding activity of HspR in a dose-dependent manner. The current study demonstrates that M. tuberculosis HrcA requires other protein(s) to function, and the heat shock protein expression in M. tuberculosis is negatively regulated jointly by HrcA and HspR.
•M. tuberculosis HrcA needs other protein partner(s) for binding the CIRCE.•Upstream element of M. tuberculosis hrcA operon has a non-conventional CIRCE for HrcA binding.•M. tuberculosis groES upstream element has both CIRCE and putative HAIR element.•HrcA also controls the expression of dnaK operon in M. tuberculosis.
Abstract only
Tuberculosis is a life threatening disease caused by the intracellular pathogen
Mycobacterium tuberculosis
(
Mtb
), resulting in around two million deaths annually worldwide.
Mtb
is ...known to have acquired certain features that help in establishing infection and persisting for longer durations in the host. Recent studies have revealed the acquisition of certain unique methyltransferases by
Mtb
as a function of phylogenetic evolution which points towards their potential involvement in mycobacterial pathogenesis. In the present study, we identified one such hypothetical methyltransferase (Rvxxxx) gene unique to
Mtb
complex i.e., absent in non‐pathogenic
Mycobacteria
. Our
in‐silico
analysis revealed that Rvxxxx gene possesses an S‐adenosylmethionine binding site and a methyltransferase domain, which was further confirmed through
in vitro s
tudies. The introduction of Rvxxxx in the genome of
Mycobacterium smegmatis
lead to significant perturbations in the physiological and morphological features of the bacilli as well as markedly enhanced its ability to resist and adapt to hostile conditions. Furthermore, recombinant
M. smegmatis
showed increase in infectivity and survival within host macrophages
ex vivo
. In addition, Rvxxxx was also found to modulate the proteome profile of recombinant
M. smegmatis
. Cumulatively, the results of this study indicate that the identified hypothetical methyltransferase Rvxxxx may play a significant role in
Mtb
pathogenesis and niche adaptation.
Support or Funding Information
The Council of Scientific and Industrial Research, Govt. of India
Human pancreatic ribonuclease (HPR) and bovine seminal ribonuclease (BS-RNase) are members of the RNase A superfamily. HPR is monomeric, whereas BS-RNase is dimeric. BS-RNase has strong antitumor and ...cytotoxic activities. However, HPR lacks cytotoxic activity as it is inactivated by intracellular cytosolic ribonuclease inhibitor (RI). Earlier, an RI-resistant cytotoxic variant of HPR, termed HPR-KNE was generated which contained three residues Lys7, Asn71 and Glu111 of HPR, known to interact with RI, mutated to alanine. In this study, we have engineered HPR to develop two dimeric RI-resistant molecules having anti-tumor activity. By incorporating two cysteines in HPR and HPR-KNE, we generated disulfide linked dimeric HPR, and a dimer of HPR-KNE, termed as HPR-D and HPR-KNE-D respectively. HPR-KNE-D was resistant towards inhibition by RI, and was found to be highly toxic to a variety of cells. On J774A.1 cells HPR-KNE-D was >375-fold more cytotoxic than HPR, and 15-fold more toxic than HPR-D. Further, on U373 cells HPR-KNE-D was >65-fold more cytotoxic than HPR, and 9-fold more toxic than HPR-D. The study demonstrates that combining dimerization and RI-resistance results in providing potent anti-tumor activity to HPR. The cytotoxic variants of HPR will be useful in designing protein therapeutics with low immunogenicity.
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